OPTICAL BIOSENSORS PRESENT AND FUTURE - PART 2 (end) pot

Three different systems will be examined: fluorophore-labeled binding proteins, FRET-based systems, and bacteria-based sensors.. Bacteria that fluoresce upon analyte binding and fluoroph

Optical Biosensors: Present and Future

F.S Ligler and C.A Rowe Taitt (editors)

9 2002 Elsevier Science B.V All fights reserved

CHAPTER 10

GENETIC ENGINEERING OF SIGNALING

MOLECULES

AGATHA FELTUS, PH.D., AND SYLVIA DAUNERT, PHARM.D., PH.D

Departments of Chemistry and Pharmaceutical Sciences

University of Kentucky Lexington, KY, USA

In order to expand the capabilities of biosensors, there is a need to develop new signaling molecules This chapter focuses on molecules, produced through genetic engineering, that combine the recognition element with a signaling element (such as a fluorophore) in an effort to optimize the signal caused by the binding o f the analyte to the recognition element These systems, while not necessarily originally developed for an optical fiber, can be immobilized at the tip of the fiber either through chemical attachment or entrapment behind a membrane Three different systems will be examined: fluorophore-labeled binding proteins, FRET-based systems, and bacteria-based sensors These systems use optical signaling methods to reveal the binding event, taking advantage of molecular biological techniques to optimize the signal The advantages and disadvantages of each system will be discussed, as well as the current state of the art of these biosensors

1 Technical Concept

In its simplest terms, a biosensor is a sensing system composed of a biological recognition element and a transducer Under the strictest definition of the term, the transducer is responsible for converting the binding event into an electrical signal Bacteria that fluoresce upon analyte binding and fluorophore-labeled binding proteins have been refered to as "reagentless biosensors" or "reagentless biosensing systems" even though they are used as assays, rather than

immobilized in a sensor There is no reason these sensing systems cannot be used as the recognition/signaling element of a fiber optic biosensor, however These systems will be discussed in both contexts, i.e., both free in solution as assays and after adapation to a fiber optic probe In order to avoid confusion, we will refer to the source of the optical signal as the signaling molecule

I.I Fluorophore-labeled binding proteins

As the name implies, a fluorophore-labeled binding protein consists of two distinct moieties: the fluorophore and the binding protein The fluorophore is covalently attached to the binding protein through the protein's amino acid side chains Binding of the analyte to the binding protein causes a conformational change in the protein structure which may result in altered optical characteristics

of the fluorophore (Figure 1)

Depending upon the actual environmental change experienced by the fluorophore, this could result in an increase or decrease in the fluorescence intensity, a change in the emission wavelength, or a change in the lifetime of the fluorophore, depending upon the characteristic to be measured These changes are generally caused by two separate phenomena: an increased or decreased polarity in the environment surrounding the fluorophore or rotational constraint

of the fluorophore For example, if the fluorophore moves from a position of high polarity (exposure to the buffer or the presence of local side chains from polar amino acids) to one of low polarity (a position inside the protein), the fluorescence will increase due to decreased quenching by the solvent molecules

or dissolved oxygen in the solvent Likewise, rotationally constraining the fluorophore's motion by trapping it inside the protein will increase fluorescence

by removing frictional energy loss caused by fluorophore movement These changes are difficult to predict beforehand and are often only revealed once the protein has actually been labeled

Having said this, even if the direction of fluorescence change cannot be predicted, knowledge of the protein structure serves as a good starting point for choosing the placement of the fluorophore From the point of view of signal-to- noise ratio, it is most advantageous to have a single fluorophore placed in a location where a large environmental change can occur Often, the most likely location for such a change is near the binding site Therefore, most initial studies are conducted by labeling at a site near the binding site as determined from observations of the crystal structure or from mutational studies

Selective labeling of the binding protein is usually accomplished via labeling through cysteine residues using sulfhydryl-selective fluorophores (For examples

of commonly employed fluorophores, see Figure 2.) In order to create one-to- one conjugates of fluorophore to protein, molecular biology is often necessary to create recombinant proteins with unique cysteine residues Using recombinant

Genetic Engineering of Signalling Molecules

Figure 1 Schematic of a fluorescently labeled protein sensing system The protein is labeled with an environmentally-sensitive fluorophore such that the binding of the analyte changes the conformation of the protein, altering the solvation of the fluorophore a) In this example, amino acid 197 of phosphate binding protein (PBP) is located near the binding pocket and will undergo a change in environment as PBP closes around its ligand, phosphate This can result in either b) an increase or c) a decrease in fluorescence upon ligand binding In some cases, the emission wavelength of the fluorophore can also change

D N A techniques, such as site-directed mutagenesis, all other cysteines in the protein are removed and other residues that will be the site of attachment are individually changed to cysteines In doing so, care must be taken not to alter any amino acids necessary for the proper functioning of the protein, such as those residues involved in analyte binding or in oligomerization of the protein This entire process will be examined in greater detail in Section 3.1

In order for this to occur, there must be a significant overlap between the emission spectrum of the donor and the excitation spectrum of the acceptor An important property of FRET is that the rate of energy transfer between the donor and the acceptor is proportional to the inverse sixth power of the distance between the two fluorophores (FRET ~ l/r6) For most pairs the F~3rster radius,

Genetic Engineering of Signalling Molecules

Figure 3 FRET-based sensing system for cAMP based on protein kinase A (PKA) This system consists of a cell line transfected with a vector coding for two fusion proteins: PKA regulatory subunit-BFP (blue fluorescent protein) and PKA catalytic subunit-GFP (green fluorescent protein) In the absence of cAMP, the regulatory and catalytic subunits associate, bringing the BFP and GFP moieties in close proximity and allowing FRET The presence of cAMP dissociates the complex of regulatory and catalytic subunits, disrupting FRET Adapted from Zaccolo et al., 2000

the distance at which the efficiency of energy transfer is 50%, is between 20A and 50/~ (Lakowicz, 1983) This distance is comparable to the size of most proteins, which allows FRET to be used when the distance between the two fluorophores will be significantly changed by the binding event This can occur

if either both fluorophores are attached to the same protein molecule and binding

of a ligand to the protein causes a conformational change that either shortens or lengthens the distance between them, or if the donor is attached to one of the binding molecules and the acceptor to the other In the latter case, a donor fluorophore attached to one of the components can transfer its energy to an acceptor fluorophore attached to the other only while the two are closely associated An example of this is given in Figure 3

FRET as a detection methodology has a number of advantages for biosenor applications Because the system employs the excitation wavelength of the acceptor and the emission wavelength of the donor, the Stokes shift is more pronounced than for fluorescence, leading to a lower background Another advantage of FRET is that the ratio of fluorescence intensities of the two

Figure 4 Schematic of a bacteria-based sensing system The bacteria are transformed with a plasmid containing the reporter gene under the control of an analyte-sensitive promoter In the presence of the analyte, the regulatory protein is released from the promoter region, allowing transcription of the reporter gene The mRNA is then translated into protein, which can be assayed The amount of protein produced is proportional to the amount of analyte present, although there is amplification at each step

so that there are many more proteins present than reporter genes Sometimes it is necessary to also place the gene for the regulatory protein on the plasmid as well as the reporter gene, as the native levels of reporter protein within the bacteria are insufficient for proper regulation of transcription

fluorophores can be used; this ratiometric technique is more accurate than measuring just one fluorescence signal

1.3 Bacteria-based sensing systems

Amplification-based methods take advantage of the high turnover of substrates to produce a large number of product molecules This is the basis of such techniques as PCR and RT-PCR In these cases, DNA or RNA molecules are selectively amplified to quantify the numbers of their parent strands Whole-cell sensing systems take this one step further by first producing DNA, which is then amplified again during the transcription to RNA, and finally amplified a third time by translation to protein

Genetic Engineering of Signalling Molecules Table 1 Reporter proteins used in whole cell sensing systems

firefly luciferase Luc

FMNI-I2 + R-CHO+ 02 -~ FMN +

BL HE0 + RCOOH + h v

coelenterazine + 02 + Ca 2+ ~

BL coelenteramide + CO2 + h t,

posttranslational formation of an internal chromophore FL

* RI, radioisotope; FL, fluorescence; CR, colorimetric; EC, electrochemical; CL,

chemiluminescence; BL, bioluminescence

A typical whole-cell sensing system consists of an organism, generally a bacterium, that is transformed with a plasmid containing a reporter gene under the control of a promoter responsive to the analyte of interest (Daunert et al., 2000; Lewis et al., 1998; Ramanathan et al., 1997a) This plasmid may also contain genes that will produce the necessary accessory proteins for the promoter, such as the regulatory proteins These additional genes are sometimes necessary, as the number of promoters on the plasmid may greatly outnumber the usual number of promoters; a larger number of regulatory proteins is necessary to regulate these plasmid-borne promoters Once the cells are exposed to analyte, transcription of the reporter gene will begin (Figure 4) After transcription, the RNA molecules are translated into protein Amplification occurs at each of these steps to produce many more protein molecules than there are reporter genes If desired, an extra level of amplification can be achieved if the reporter protein is

an enzyme that will turn over large numbers of substrate molecules However, if this further amplification is not required, then a protein such as the green fluorescent protein (GFP) can be used GFP does not require addition of an external substrate, as the protein itself emits green fluorescence upon excitation

at 490 nm Another way to obviate adding a substrate is to use the entire lux cassette, instead of just luxAB, to produce bacterial luciferase In this way all the accessory proteins to produce the substrates necessary for bacterial luciferase activity are also transcribed (Manen et al., 1997)

The sensitivity of these systems is determined by a number of factors The response of the promoter must be taken into account, but the largest effect of the promoter/repressor protein is upon the selectivity of the system The more controllable factor is the choice of reporter protein, since there is often only a limited choice of promoters for a given analyte The ideal reporter protein will

be easy to use, have an easily discernable signal over the background, and have a wide dynamic range and high sensitivity (Daunert et al., 2000) Examples of reporter proteins that have been used to develop whole-cell sensing systems are given in Table 1 Sensitivity can be a function of several factors, including the detection method, efficiency of expression, reporter protein turnover number (if the protein is an enzyme), and, if applicable, the endogeneous levels of the reporter protein For this reason, bioluminescent reporter proteins are a popular choice because bioluminescence is not found in most organisms, and is a very sensitive method of detection

2 History

These three types of fluorescent signaling systems emerged from the need of researchers in the biological sciences to study protein response to the binding of various ligands For example, bacteria-based sensing systems are the result of experiments on regulation of transcription at various promoters

2.1 Fluorophore-labeled proteins and FRET-based systems

These two systems share a common ancestry in studies of protein function One way of examining the structural changes in proteins upon ligand binding, dimerization, or denaturation is by measuring in the native fluorescence of tryptophan residues This approach since they might not be close can be used to measure binding only when the tryptophan is proximal to the active site This limitation led to the use of fluorescent cofactors and substrates, such as flavin mononucleotide, to study changes occurring within the binding pocket Later, proteins were labeled with extrinsic fluorophores Such labeled proteins have been used for a number of applications, including microinjection into cells to study protein localization and solution studies of protein structural changes Initially, biochemists used these fluorophore-labeled proteins to gain information about the alterations in size, shape, and binding properties of proteins However, with the development of environmentally sensitive fluorophores and the ability to produce mutated recombinant proteins, the fluorophore-labeled sensing system as

it stands today was born Table 2 gives several examples of analytes that have been measured using these systems Most of the currently-developed sensing systems of this type depend upon molecular biology to either create a unique site for fluorophore attachment, to translate the protein such that it incorporates non- native fluorescent amino acids, or to fuse a GFP to the protein

Genetic Engineering of Signalling Molecules

Table 2 Examples of fluorophore-labeled protein sensing systems In vitro/in vivo refers to whether the protein is used in situ after being produced by the cells or whether the proteins are expressed, isolated, and purified prior to use, and used as a sensing system

.

fatty acids I-FABP In vitro Richieri et al., 1992

biotin Streptavidin In vitro Murakami et al., 2000 Ca2+ CaM-YFP In vitro~in Baird et al., 1999

Thompson et al., 1998 Salins et al., 2001;

Tolosa et al., 1999 Dattelbaum and

Lakowicz, 2001

*Abbreviations: PBP, phosphate binding protein; I-FABP, intestinal fatty acid binding protein; MBP, maltose binding protein; CaM, calmodulin; YFP, yellow fluorescent protein; EGFP, enhanced green fluorescent protein; GGBP, galactose/glucose-binding protein; GlnBP, glutamine binding protein

FRET-based systems can be considered as a subclass of the fluorophore-labeled proteins, different only because they depend upon the proteins being labeled with two fluorophores rather than one Because FRET is a distance-dependent phenomenon, it was originally used to study assembly of multi-subunit protein complexes, such as ribosomes, or interaction between a protein and cellular membranes In the 1990s, however, FRET-based systems started to be used for analytical purposes (Table 3) The most recent trend is to use GFP and its wavelength-shifted mutants as the donor and acceptor molecules

Table 3 FRET-based assays using labeled proteins.*

factor Xa factor Xa site " BFP rsGfp Mitra et ai.',' 1996 caspase-3 Caspase-3 site BFP GFP Xu et., 1998

caspase-3 Caspase-3 site CFP YFP Jones et al., 2000

Zn 2+

Ca 2+

zinc finger Lissamine rhodamin Godwin and Ber,

aequorin Aequorin GFP Baubet et al., 2000

Ca 2+

PKA

Cam/M13 BFP/CFP GFP/YFP Miyawaki et al.,

1997

CAMP PKA fluorescei rhodamin Adams et al., 1991

*Abbreviations: BFP, blue fluorescent protein; GFP, green fluorescent protein; CFP, cyan fluorescent protein; YFP, yellow fluorescent protein; CaM, calmodulin; PKA, protein kinase A; KID, kinase inducible domain

Because these fluorophores are proteins themselves, plasmid constructs can be made that fuse the G F P to the sensing protein, allowing these proteins to be

produced within a cell and used in situ as sensors This is advantageous, since

the analytes of interest are generally intracellular second messengers Moreover, the need to microinject purified chemically-labeled proteins is avoided

2.2 Bacteria-based sensing systems

Sensing systems of this type trace their origin back to bioassays in which nutrient-deficient or antibiotic-resistant strains were plated on media containing various concentrations of the analyte and the surviving number of cells counted

F r o m this methodology evolved the non-specific bacteria-based sensing systems These bacteria constitutively express a reporter protein while alive, but as toxins begin tokill the bacteria, the protein is no longer produced, giving a lower signal

At the same time, the bacterial operons were discovered, and reporter genes were

Genetic Engineering of Signalling Molecules Table 4 Promoters used to develop whole-cell sensing systems

Corbisier et al., 1999 Virta et al., 1995 Sticher et al., 1997

de Lorenzo et al., 1993; Ikariyama et al., 1997; Willardson et al., 1998

Selifonova and Eaton, 1996 Heitzer et al., 1994; King et al., 1990 Guan et al., 2000

Shetty et al., 1999 ~ Daunert et al., 2000; Shrestha et al., 2000 Billinton et al., 1998; Ptitsyn et al., 1997; Rettberg et al., 1997, 1999; van der Lelie

et al., 1997

placed under their control in order to study gene expression The first of the bacteria-based sensing systems for specific analytes used metal- or toxin- resistance promoters, presumably because these operons are generally carried on plasmids rather than within the bacterial genome and because expression is tightly regulated and induction occurs in response to the presence of the toxin or metal Since then, other analytes have been targeted, including sugars (Table 4) Because fluorescent report proteins have been shown to work well, the newest trend is to use fluorescent reporter proteins, rather than bioluminescent ones,

because fluorescent proteins require no addition of substrates There is also a need to develop sensing strains that can respond to more than one analyte This has been accomplished by using two separate promoters and reporter proteins

3 State of the Art

In the previous section we discussed the history of how the fluorescent signaling systems emerged In this section, we Will focus on the more advanced forms of these systems, and describe particular sensing systems in detail to give the reader

an idea of the full scope of the system, some considerations of how the system is designed, and the part that molecular biology plays in it

3.1 Fluorophore-labeled binding proteins

This particular mechanism has been exploited to develop a number of fluorescence assays for a variety of analytes (Table 2) Molecular biology is often employed in order to control the site of fluorophore attachment to give the greatest change upon ligand binding In several cases, genetic engineering was employed to introduce a unique cysteine in the binding protein This residue was then specifically labeled with a thiol-reactive environmentally-sensitive fluorescent probe (Brune et al., 1998; Gilardi et al., 1994; Salins et al., 1998; Schauer-Vukasinovic et al., 1997; Thompson et al., 1998) In the case of I- FABP, this was unnecessary as acrylodan reacted only at lysine 27 and produced large changes in fluorescence upon addition of free fatty acids such as oleate, palmitate, and arachidonate (Richieri et al., 1992) Murakami et al (2000) used a different approach, introducing a unique L-2-anthrylalanine into the amino acid

sequence of streptavidin by an in vitro transcription method, thus creating a

sensing system for biotin

The optimal site of fluorophore location can be determined through examination

of the crystal structure of the protein or through NMR studies of the free and bound forms of the protein If these have not been determined, then an educated guess can be made through other studies, such as mutagenesis to determine the location of the analyte binding site or through examination of a closely related protein This protein engineering strategy allows for the specific attachment of a fluorophore to the protein at a site which undergoes a large change in its environment upon ligand binding In some cases, it may be necessary to try several sites and fluorophores before the optimal response of the system can be established

The development of the sensing system for C a 2§ based on calmodulin (CAM) by Schauer-Vukashinovic et al (1997) is a good example The protein calmodulin binds four calcium ions located in pairs in each of two domains: an N-terminal domain and a C-terminal domain that are linked by a long helix (Babu et al.,

Genetic Engineering of Signalling Molecules

1988; Kuboniwa et al., 1995) In the absence of calcium, the structure is more disordered When calcium binds, two hydrophobic pockets open, one in each domain, for binding to proteins such as myosin light chain kinase (MLCK) or to drugs such as trifluoropiperazine and phenothiazine Figure 5 shows a close-up view of the C-terminal binding pocket in both the presence and absence of calcium (Finn et al., 1993) Several mutants of CaM were produced with unique cysteine residues at positions 38, 81,109, and 113 (Schauer-Vukashinovic et al., 1997) The last three are seen in Figure 5 near the pocket; residue 38 is in the N- terminal domain Several combinations of thiol-reactive fluorophores and labeling sites were examined The best results were obtained with a CaM109- MDCC conjugate (96% increase in fluorescence upon Ca 2+ binding) When the other sites were labeled with MDCC, the amount of increase was only 15%, 16%, and 28%, respectively As seen in Figure 5, residues 81, 109, and 113 are located quite close to each other in the structure of CaM, but the three residues apparently have very different environmental changes upon Ca 2+ binding There are also differences when the fluorophore at position 109 is exchanged for another If, instead of MDCC, the related fluorophore CPM (Figure 2) is used, the amount of change is only 25 % If fluorescein is used, then there is no change

in signal upon Ca 2+ binding

The limit for detection of calcium using CaM109-MDCC is 2 x 10 -9 M C a 2+ A random labeling at lysine residues (of which CaM has 9) with fluorescein isothiocyanate shows a lower amount of change in fluorescence (23% increase upon Ca 2+ binding) and a higher detection limit (5 x 10 8 M) (Blair et al., 1994) The reason is that the change in fluorescence is highly dependent upon the location of the fluorophore within the protein Nonspecific labeling with multiple fluorophores increases the background signal, giving a smaller relative increase in fluorescence upon calcium binding This reduces the ability to detect lower levels of Ca 2§ Similar effects have been seen in the systems for maltose (Gilardi et al., 1994) and phosphate (Brune et al., 1994), indicating that the best detection limits are obtained when a unique fluorophore is properly positioned

An alternate strategy has recently been employed using GFP instead of a small organic fluorophores These studies use circular permutations of GFP (cpGFP); the C-terminus is fused to the N-terminus Baird et al (1999) inserted CaM into

a circular YFP at amino acid 145 The fluorescence of the YFP was retained while giving rise to a Ca2§ fusion protein with a detection limit of approximately 2 x 10 "6 M C a 2+ Nakai et al (2001) used a similar construct in which GFP was circularized to produce a new N-terminus at residue 149 and C- terminus at residue 144 The calmodulin-binding peptide M13 was attached to the new N-terminus and CaM to the new C-terminus The resulting protein showed an increase in fluorescence of up to 4.5-fold upon addition of Ca 2+ because the CaM moiety bound the M13 moiety, altering the conformation of GFP The detection limit for this system was one order of magnitude better

Figure 5 Alterations in the C-terminal hydrophobic pocket of calmodulin upon calcium binding Residue 109 is closer to the pocket than either residue 81 or 113 Labeling mutant calmodulins gives the most change with an MDCC-CaM109 conjugate Labeling

at 81 or 113 does not give as much fluorescence change upon calcium binding, presumably because the two residues are further from the hydrophobic pocket than amino acid 109 Adapted from Schauer-Vukasinovic et al (1997)

(1 x 10 "7 M) than the system developed by Baird et al., although the Nakai system has a narrower dynamic range Both systems were also shown to be useful in

detecting calcium fluxes in vivo (Baird et al., 1999; Nakai et al., 2001)

3.2 FRET-based systems

FRET systems have been used to detect analytes and biological functions as varied as protease activity, ions, cyclic AMP, myosin II phosphorylation, and insulin-receptor signaling As seen in Table 3, these assays can either be

performed in vitro or in vivo by microinjection or transfection with genes to transcribe the sensing systems in situ Molecular biology is used to create the

GFP or other fusion proteins necessary for each sensor For biosensing purposes,

these labeled proteins can first be produced in vivo, then purified and

immobilized at the tip of a fiber optic probe It is also possible that the cells themselves could be immobilized, obviating the purification step

One of the most important contributions of molecular biology to these systems has been the creation of GFP mutants that can act as FRET pairs Native

Aequorea GFP absorbs blue light and emits green light Its usual FRET donor is

the blue fluorescent protein (BFP), a variant of GFP mutated in several residues

320

Genetic Engineering of Signalling Molecules

Figure 6 FRET-based sensing system for Ca 2§ based on a BFP/GFP pair bridged with a MLCK CaM binding site The FRET donor BFP is separated from the acceptor GFP by a CaM recognition sequence from myosin light chain kinase (MLCK) CaM can only bind this sequence in the presence of Ca 2§ increasing the distance between the fluorophores and decreasing the amount of FRET The system, therefore, responds to the amount of

Ca 2§ present Adapted from Miyawaki et al (1997)

in and around the chromophore of the protein; these changes shift the excitation

to UV wavelengths and the emission to blue This provides a spectral overlap with GFP, allowing FRET The other FRET pair used, cyan fluorescent protein (CFP, donor) and yellow fluorescent protein (YFP, acceptor), is composed of two mutant GFPs created in the same way In this pair, CFP absorbs in the blue region and emits in the blue-green region, overlapping with YFP's absorption spectrum YFP then emits in the yellow A discussion of the many different mutants of GFP can be found in a review paper by Tsien (1998)

The assays of protease activity do not depend upon a binding event, but rather the physical separation of tethered fluorophores In these systems, EGFP and BFP are connected by a short peptide sequence containing a cleavage site for the protease of interest Before the protease acts at its site, EGFP and BFP are kept

at a fixed distance from each other; cleaving the bond within the cut site causes the fluorophores to drift apart, thus disrupting the FRET For trypsin, the amount

of change in the fluorescence emission ratio was 4.6-fold and for factor Xa it was 3-fold (Mitra et al., 1996) Since EGFP, the linker, and BFP are all genetically encoded, an in vivo assay can be developed by transfecting cells with DNA to produce the sensor inside the cells This was demonstrated by Xu et al (1998) in their system for caspase-3 Activation of caspase-3 destroyed FRET between the GFPs Such assays are of particular value in the high-throughput screening of apoptosis-inducing drugs, since caspase-3 is activated during apoptosis Indeed, recently Jones et al (2000) reported that this system could reliably identify apoptosis-inducing drugs, such as staurosporine, camptothecin, and etoposide Cell signaling events, such as Zn 2§ or Ca 2§ release and cAMP accumulation, have also been found to be good targets for FRET-based systems An in vitro system

321

for Zn 2§ developed by Godwin and Berg (1996) uses a zinc finger peptide as the sensing element Zinc fingers bind zinc tightly and have a great selectivity for Zn(II) over Co(II), Fe(II), and Ni(II) Godwin and Berg (1996) engineered a zinc finger with a lissamine donor at the N-terminus and a fluorescein acceptor at the C-terminus Binding of Zn 2§ to the peptide brings together the two fluorophores, resulting in FRET This system has the ability to detect Zn 2+ at levels of 5 x 10 -7

M (Godwin and Berg, 1996) Ramoser et al (1997) developed a sensing system for Ca ~+ by connecting two GFP variants, BFP and RGFP, with a peptide linker containing the calmodulin binding sequence from myosin light chain kinase Binding of (Ca2+)4-CaM to the sensor increases the inter-fluorophore distance from ~25/~ to ~65A, effectively eliminating FRET (Figure 6) The change in the fluorescence emission ratio is dose-dependent for both Ca 2§ and (Ca2+)4-CaM and

is shown to work well when microinjected into cells, as well as in vitro

A similar system was developed by Miyawaki et al (1997) using BFP or CFP, CaM, CaM-binding peptide M13, and GFP Binding of Ca 2+ causes the CaM moiety to wrap around the M13 peptide, decreasing the distance between the pairs and aligning them properly for FRET This results in a 70% increase in the fluorescence emission ratio and a wide detection range of three orders of magnitude from 10 -7 tO 10 -4 M , with a detection limit of 2.5 x 10 -8 M Ca 2§ By transfecting the DNA for this sensor into mammalian cells, it was found that a different pair of GFPs (CFP and YFP) worked better by improving the brightness (CFP fluoresces more intensely than BFP) and signal-to-noise ratio However, the overall amount of change was only 1.5-fold for the CFP/BFP pair versus 1.8- fold for the BFP/GFP pair, due to bleedthrough of CFP emission into the YFP spectrum

A bridged GFP chimera has also been developed for sensing of cAMP-related effects in cells Nagai et al (2000) created a sensor based on a bridge of kinase- inducible domain of CREB (cAMP response element binding protein) This domain is phosphorylated by cAMP-dependent protein kinase A (PKA), which results in a conformational change BFP and RGFP were again used as the donor and acceptor, located at the two ends of the bridge In vitro experiments with this system showed that the emission ratio increased from 0.68 to 0.83 when incubated with PKA and ATP Transfection of the chimera into COS-7 cells showed an increase in fluorescence upon PKA activation while administration of PKA inhibitor H-89 significantly inhibited FRET within the cells

Cyclic AMP itself has been monitored in vivo using fluorescently-tagged PKA PKA consists of regulatory and catalytic subunits that dissociate upon cAMP binding This disrupts FRET between the donor on the regulatory subunit and the acceptor on the catalytic subunit (Figure 2) The original sensor developed

by Adams et al (1991) used a fluorescein/rhodamine pair This sensor worked very well, but required expression and purification of the protein subunits, in

Genetic Engineering of Signalling Molecules

a completely in vivo system using BFP and GFP (Figure 2) In a population of transfected COS-7 cells treated with 10 #M isoproterenol, the emission ratio increased from 1.7 to 2.0, and was completely reversed by incubation with 10

#M propranolol This reaction is almost instantaneous upon introduction of isoproterenol These systems thus offer an extremely fast response to their analytes

3.3 Bacteria-based sensing systems

Bacteria-based sensing systems have been developed for a variety of analytes

As shown in Table 4, there are a number of promoters that have been used for either the specific sensing of a particular analyte, a family of compounds, or a stress response, such as starvation Many are used to detect toxic substances, such as heavy metals, carcinogens, or organic pollutants For example, sensing systems have been developed for arsenic/antimony (Corbisier et al., 1993; Ramanathan et al., 1997b, 1998; Scott et al., 1997; Tauriainen et al., 1997), copper (Corbisier et al., 1999; Shetty et al., 2000), cadmium/lead (Corbisier et al., 1999; Tauriainen et al., 1998), chromium (Peitzsch et al., 1998), aluminum (Guzzo et al., 1992), and mercury (Virta et al., 1995) The original purpose of these promoters is to produce proteins that either sequester the metal ions, transport them outside the cell, or enzymatically detoxify them (Brown et al., 1998; Nies, 1999) Experience has shown that these systems are extremely sensitive to very small amounts of the metal being present In fact, in one case,

by coupling the ars promoter with the gene coding for bacterial luciferase, Ramanathan et al (1997b) found that a detection limit of 10 ~5 M arsenite could

be obtained with high selectivity for antimonite and arsenite over other metals such as bismuth, cadmium, and cobalt

High selectivity was also seen in a sensing system for L-arabinose developed using the PBAD promoter and the gene for GFP developed by Shetty et al (1999)

In cases of low glucose levels, E coli can use other sugars as an energy source This system could detect 1 x 10 "7 M L-arabinose while it did not respond to other pentose sugars or their corresponding D-isomers These bacteria were also immobilized at the tip of a fiber optic A small sleeve was placed over the tip of the fiber optic, creating a small space in which the bacterial suspension was kept The opening was covered with a dialysis membrane to prevent the bacteria from diffusing out of the sensing range of the fiber optic, but still allow the analyte to pass through and interact with the bacteria The sensor had a detection limit one order of magnitude less sensitive than the solution-based system; this decrease in sensitivity was attributed to changes in the instrumental setup (i.e., a lower- powered light source, decreased coupling efficiency, a less sensitive PMT, and an increased diffusion time) Another study using this system showed the probable direction that these systems will take in the future A dual-detection system for L-arabinose and 13-lactose was developed by combining the arabinose system described above with a similar system to detect lactose (Daunert et al., 2000;

Shrestha et al., 2000) The lactose system employed the gene for BFP, which emits in the blue region Thus, two analytes could be measured at the same time

by simultaneously monitoring the fluorescence emission at two different wavelengths

In order to survive in heavily polluted environments, certain organisms have also developed the capability of using organic pollutants as carbon sources Promoters from operons metabolizing these environmental pollutants have been used to develop biosensing systems for the monitoring of the bioavailable amounts of chemicals such as alkanes (Sticher et al., 1997), benzene derivatives (de Lorenzo et al., 1993; Ikariyama et al., 1997; Selifonova and Eaton, 1996; Willardson et al., 1998), chlorocatechols (Guan et al., 2000), and PCBs (Layton

et al., 1998) Likewise, sensing systems for carcinogens, such as the SOS-lux system, are capable of monitoring genotoxins by responding to actual DNA

damage of the cda promoter by the environmental toxin by producing bacterial

luciferase in a dose-dependent manner (Ptitsyn et al., 1997; Rettberg et al., 1997)

It is important to note that this system responds not to the concentration of the carcinogen within the cell, but its activity The SOS-/ux system also has advantages over the Ames test in that results are available within 1-2 h and kinetic effects of the toxin can be studied

4 Advantages and Limitations

Of the systems described in this chapter, the two with the fastest response times are the binding protein-based systems Compared to FRET-based probes, fluorophore-labeled binding proteins usually have greater fluorescence changes, presumably because they depend upon a direct action upon the fluorophore rather than on the more indirect method of energy transfer Also, because these systems usually are based on using single-chain proteins, they are capable of being covalently immobilized on a solid surface This is more difficult in FRET-based systems because the two component proteins must be free to interact with or dissociate from one another

Despite these advantages, fluorophore-labeled binding proteins are more difficult

to optimize, as several different immobilization sites and fluorophores must be tested This represents a substantial amount of molecular biology, and can take quite a long time The main reason for this is the difficulty in predicting the environmental change at a specific location on the protein Although clues can

be obtained from crystallographic and NMR structures, even small changes in location make a very large difference to the sensitivity of the system It is much easier to predict whether the distance between two fluorophores will change upon the binding of the analyte

Genetic Engineering of Signalling Molecules

One of the main advantages of FRET-based sensing systems is that they employ

a very small number of reagents In fact, in some cases they use no additional reagents, as both the fluorophores (GFP, YFP, etc.) and the binding proteins can

be genetically encoded and produced within the cells Like the whole-cell based systems, they may be extremely cost-effective, since the transformed cells can be frozen and a new batch of sensitive cells regrown at any time

FRET as a detection methodology has additional advantages that make it attractive for use in biological systems Because the system uses the excitation wavelength of the acceptor and the emission wavelength of the donor, the Stokes shift is more pronounced than for fluorescence, resulting in a lower background Another advantage of FRET is that the ratio of fluorescence intensities can be used; this technique is more accurate than measuring a single fluorescence intensity Ratiometric methods are also independent of path-length, accessible volume, and local concentration, points that become more important as we consider decreasing the assay volume (Giuliano and Taylor, 1998) Having said this, it is not always possible to develop a FPdET-based system due to the necessity of having some sort of change in distance occur between the fluorophores Also, because of the association/dissociation of the protein components, FRET may, in some cases, be more susceptible to matrix effects if some component of the sample causes premature dissociation

The least susceptible system to matrix effects is probably the whole-cell based sensing system In order for transcription activation to occur, the analyte must be taken up by the bacteria and then interact at the promoter to induce expression of the reporter gene Not only is it unlikely that an interferent will mimic these steps, but the bacterial cell wall gives the bacteria a high tolerance to pH changes and to other environmental extremes A high level of selectivity is also found in these systems for the same reasons; the interfering species must not only be able

to enter the cell, but it must cause the proper conformational changes in the promoter's regulatory protein to initiate transcription Another advantage is the improved sensitivity of the system due to the number of amplification steps The amount of molecular biology required to develop a whole-cell sensing system is less than for either of the protein-based systems described above, as there is no need to mutate the reporter protein In addition, the system is continuously renewable If a new batch is needed, it is simply regrown; no purification is necessary The main disadvantage of these systems is the long response times Because the bacteria must be alive and growing, it may be necessary to incubate the ceils at 37~ for several hours in order to take up the analyte and produce a properly-folded reporter protein Protein-based systems,

on the other hand, bypass this step and require only minutes of incubation Notwithstanding the more extended time requirement, the bacteria-based systems are limited only by the ability to find a promoter that responds to an analyte of interest With proper choice of reporter gene and detection method, highly sensitive and selective systems can be developed to study not only the

concentrations of various analytes, but, more importantly, to study their actual activities

5 Potential for Expanding Current Capabilities

The range of analytes that can be measured using the systems described in this chapter is limited only by the availability of recognition elements For example, ' the selectivity of bacteria-based systems is controlled by the selectivity of the binding proteins regulating the promoter's activity Not only is it possible that new promoters will be discovered, but that by mutation and selection of bacterial strains, new promoters can be created, as they have been naturally over the course of time by new stresses placed on microorganisms living in harsh or nutrient-depleted environments New binding proteins as the basis of FRET- or fluorophore-labeled systems can be created through random synthesis or DNA shuffling These can serve to either create binding proteins for new analytes or to increase the selectivity or binding affinity of known binding proteins

FRET-based systems depending upon two GFPs as the donor and acceptor molecules may also be improved through the creation of new GFP variants In the past, there has been a focus on creating GFPs that are brighter, have higher quantum yields, are more stable, and have different absorption and emission wavelengths than the wild-type protein (Tsien, 1998) This approach also has the potential to expand whole-cell sensing systems into the multi-analyte area Another area in which these systems can find use is in small-volume analyses Many high-throughout screening (HTS) applications are beginning to take advantage of advances in microfluidics and microfabrication to shrink the size of assays Since FRET-based systems are already performed in vivo and observed

in single cells, they are already proven to be applicable to small volumes Fluorophore-labeled binding proteins could also be of use, not only in small volumes and microfluidic platforms, where decreasing the number of aliquots will decrease the error, but also in single cells, where injection of a very limited number of assay components is necessary to prevent the cell from bursting In time, ways will be found that obviate these microinjections, so that the sensing protein will be transcribed in situ within the cells, as FRET-based systems are today This will further expand their use in biological systems

Genetic Engineering of Signalling Molecules

Baird, G.S., D.A Zacharias and R.Y Tsien, 1999, Proc Natl Acad Sci U S A

96, 11241

Baubet, V., H Le Mouellic, A.K Campbell, E Lucas-Meunier, P Fossier and P

Brulet, 2000, Proc Natl Acad Sci U.S.A 97, 7260

Billinton, N., M.G Barker, C.E Michel, A.W Knight, W.D Heyer, N.J

Goddard, P.R Fielden and R.M Walmsley, 1998, Biosens Bioelectron

13, 831

Blair, T.L., S.-T Yang, T Smith-Palmer and L.G Bachas, 1994, Anal Chem

66, 300

Brown, N.L., J.R Lloyd, K Jakeman, J.L Hobman, I Bontidean, B Mattiasson

and E Csoregi, 1998, B iochem Soc Trans 26, 662

Brune, M., J.L Hunter, J.E Corrie and M.R Webb, 1994, Biochemistry 33,

Corbisier, P., D van der Lelie, B Borremans, A Provoost, V de Lorenzo, N.L

Brown, J.R Lloyd, J.L Hobman, E Csoregi, G Johansson and B Mattiasson, 1999, Anal Chim Acta 387, 235

Dattelbaum, J.D and J.R Lakowicz, 2001, Anal Biochem 291, 89

Daunert, S., G Barrett, J.S Feliciano, R.S Shetty, S Shrestha and W Smith-

Spencer, 2000, Chem Rev 100, 2705

de Lorenzo, V., S Fernandez, M Herrero, U Jakubzik and K.N Timmis, 1993,

Gene 130, 41

Finn, B.E., T Drakenberg and S Forsen, 1993, FEBS Lett 336, 368

Gilardi, G., L.Q Zhou, L Hibbert and A.E Cass, 1994, Anal Chem 66, 3840 Giuliano, K.A and P.L Post, 1995, Annu Rev Biophys Biomol Struct 24,

405

Giuliano, K.A and D.L Taylor, 1998, Trends Biotechnol 16, 135

Godwin, H.A and J.M Berg, 1996, J Amer Chem Soc 118, 6514

Guan, X., S Ramanathan, J.P Garris, R.S Shetty, C.M Ensor, L.G Bachas and

S Daunert, 2000, Anal Chem 2423

Guzzo, J., A Guzzo and M.S DuBow, 1992, Toxicol Lett 64-65 Spec No, 687 Heitzer, A., K Malachowsky, J.E Thonnard, P.R Bienkowski, D.C White and

G.S Sayler, 1994, Appl Environ Microbiol 60, 1487

Ikariyama, Y., S Nishiguchi, T Koyama, E Kobatake, M Aizawa, M Tsuda

and T Nakazawa, 1997, Anal Chem 69, 2600

Jones, J., R Heim, E Hare, J Stack and B.A Pollok, 2000, J Biomol Screen 5,

307

King, J.M.H., P.M Digrazia, B Applegate, R Burlage, J Sanseverino, P

Dunbar, F Larimer and G.S Sayler, 1990, Science 249, 778

Kuboniwa, H., N Tjandra, S Grzesiek, H Ren, C.B Klee and A Bax, 1995,

Nature Struct Biol 2, 768

327

Lakowicz, J.R., 1983, Principles of Fluorescence Spectroscopy: Energy Transfer,

Eds Academic Press, New York

Layton, A.C., M Muccini, M.M Ghosh and G.S Sayler, 1998, Appl Environ

Miyawaki, A., J Llopis, R Heim, J.M McCaffery, J.A Adams, M Ikura and

R.Y Tsien, 1997, Nature 388, 882

Murakami, H., T Hohsaka, Y Ashizuka, K Hashimoto and M Sisido, 2000,

Biomacromolecules 1, 118

Nagai, Y., M Miyazaki, R Aoki, T Zama, S Inouye, K Hirose, M fino and M

Hagiwara, 2000, Nature Biotechnol 18, 313

Nakai, J., M Ohkura and K Imoto, 2001, Nature Biotechnol 19, 137

Nies, D.H., 1999, Appl Microbiol Biotechnol 51,730

Peitzsch, N., G Eberz and D.H Nies, 1998, Appl Environ Microbiol 64, 453 Ptitsyn, L.R., G Horneck, O Komova, S Kozubek, E.A Krasavin, M Bonev

and P Rettberg, 1997, Appl Environ Microbiol 63, 4377

Ramanathan, S., M Ensor and S Daunert, 1997a, Trends Biotechnol 15,500 Ramanathan, S., W Shi, B.P Rosen and S Daunert, 1997b, Anal Chem 69,

3380

Ramanathan, S., W Shi, B.P Rosen and S Daunert, 1998, Anal Chim Acta

369, 189

Rettberg, P., L.R Ptitsyn, O Komova, S Kozubek, E Krasavin, M Bonev and

G Homeck, 1997, Mut Res 379, $206

Rettberg, P., C Baumstark-Khan, K Bandel, L.R Ptitsyn and G Horneck, 1999,

Anal Chim Acta 387, 289

Richieri, G.V., R.T Ogata and A.M Kleinfeld, 1992, J Biol Chem 267, 23495 Romoser, V.A., P.M Hinkle and A Persechini, 1997, J Biol Chem 272, 13270 Salins, L.L.E., V Schauer-Vukasinovic and S Daunert, 1998, Proc SPIE 3270,

Selifonova, O.V and R.W Eaton, 1996, Appl Envir Microbiol 62, 778

Shetty, R.S., Y Liu, S Ramanathan and S Daunert, 2000, The Pittsburgh

Conference on Analytical Chemistry Applied Spectrometry, New Orleans, LA

Shetty, R.S., S Ramanathan, I.H Badr, J.L Wolford and S Daunert, 1999, Anal

Chem 71,763

Genetic Engineering of Signalling Molecules

Shrestha, S., R.S Shetty, S Ramanathan and S Daunert, 2000, 219th Meeting of

the American Chemical Society, San Francisco, CA, ANYL-047

Sticher, P., M.C Jaspers, K Stemmler, H Harms, A.J Zehnder and J.R van der

Meer, 1997, Appl Environ Microbiol 63, 4053

Tauriainen, S., M Karp, W Chang and M Virta, 1997, Appl Environ

Tolosa, L., I Gryczynski, L.R Eichhom, J.D Dattelbaum, F.N Castellano, G

Rao and J.R Lakowicz, 1999, Anal B iochem 267, 114

Tsien, R.Y., 1998, Ann Rev Biochem 67, 509

van der Lelie, D., L Regniers, B Borremans, A Provoost and L Verschaeve,

1997, Mutat Res 389, 279

Virta, M., J Lampinen and M Karp, 1995, Anal Chem 67, 667

Willardson, B.M., J.F Wilkins, T.A Rand, J.M Schupp, K.K Hill, P Keim and

P.J Jackson, 1998, Appl Environ Microbiol 64, 1006

Xu, X., A.L Gerard, B.C Huang, D.C Anderson, D.G Payan and Y Luo, 1998,

Nucleic Acids Res 26, 2034

Zaccolo, M., F De Giorgi, C.Y Cho, L Feng, T Knapp, P.A Negulescu, S.S

Taylor, R.Y Tsien and T Pozzan, 2000, Nature Cell Biol 2, 25

F.S Ligler and C.A Rowe Taitt (editors)

9 2002 Elsevier Science B.V All fights reserved

CHAPTER 11

ARTIFICIAL RECEPTORS FOR CHEMOSENSORS

THOMAS W BELL, PH.D AND NICHOLAS M HEXT, PH.D

Department of Chemistry, University of Nevada, Reno

Reno, NV 89557-0020 U S A

Chemosensors are molecules of abiotic origin that signal the presence of matter which can be used to measure the concentrations of analytes in solution They consist of artificial receptors tailored to reversibly bind the analyte with sufficient affinity and selectivity, a chromophore or fluorophore, and a mechanism for communicating between binding and optical signaling This chapter details chemosensor design considerations, gives historical background, and provides examples of chemosensors for neutral organic molecules and various anions Chemosensors for biologically important analytes are particularly emphasized

I Technical Concept

Sensors for solutes found in low concentration, as is typically the case for samples of biological or environmental origin, generally require binding or concentration of the analyte by the sensor for adequate sensitivity Our ability to develop sensors for new analytes is often limited by the paucity of materials having adequate affinity, as well as selectivity, when the latter is needed to distinguish the analyte from interfering substances Enzyme-based biosensors are restricted to the detection of naturally occurring substrates and cofactors Major advances are being made in adapting biomolecules, such as antibodies and aptamers, for sensor applications, but artificial receptors have many potential advantages

Because they are created by enzymatic chemical reactions, biotic receptors are composed of a limited range of molecular subunits, including amino acids, nucleotides, and sugars The analyte binding site is generally produced by

Guest

Host

Bell and Hext

Figure 1 Cartoon showing binding ofan analyte (guest) by a chemosensor (host), producing a complex with altered optical properties, here an increase in fluorescence

secondary interactions between subunits located along a linear chain Thus, the critical ability of a biosensor to selectively bind the analyte can be destroyed by variations in ambient conditions, including pH, oxidizing agents, and heat, causing either chemical or thermal degradation, or denaturation

Abiotic receptors can be synthesized from chemically robust components and the binding site can consist of a cavity or cleft enforced by stable, covalent bonds Their molecular architectures are limited only by the capabilities of synthetic organic chemistry, not by the range of substructures accepted as enzyme substrates Hence, artificial receptors can be tailored for an unlimited variety of analytes Their affinities, optical properties, solubilities, and other important characteristics can be adjusted to meet requisite sensor specifications

1.1 Chemosensor design

information into analytically useful signals (Hulanicki et al., 1991) The term

presence of matter or energy (Czamik, 1993a) Indeed, molecules can be thought

of as miniscule devices that can be engineered, fabricated, and used to perform useful functions Analyte binding can induce mechanical motion (conformational change) in molecules, leading some to term chemosensors operating in this way "molecular machines" (Shinkai et al., 2000; Pina et al., 2000), a category of molecular devices that currently is of intense interest (Balzani et al., 2000; Sauvage, 2001) Let's now examine how chemosensors work and what factors must be considered during chemosensor design

A key requirement of chemosensor function is that analyte binding must occur

equilibrium by optical detection of either the chemosensor-bound species or the analyte-free chemosensor It also permits continuous measurements to be made with dynamic optical response to changing analyte concentrations Irreversible chemical reactions produce chemodosimeters (Czarnik, 1993a), which can

measure cumulative amounts of reactants Such reactions are useful in single- measurement applications, as with timed-response uses of disposable sensors Here we deal only with the more broadly applicable phenomena of reversible association or reversible chemical reaction between the chemosensor and its analyte

A chemosensor consists of a molecule incorporating a binding site, a chromophore or fluorophore, and a mechanism for communication between the two (Czamik, 1994) Analyte binding thus produces a change in chemosensor optical properties (absorption or fluorescence), as illustrated in Figure 1 Expressed in terms of host/guest chemistry (Cram and Cram, 1978; Cram, 1988), the position of the equilibrium established between the host (chemosensor), its guest (analyte) and the complex (or reversible reaction product) is governed by the association constant (Ka) as described by Equations 1 and 2

in guest concentration may not produce sufficiently large changes in the optical signal Thus, a key performance characteristic of a chemosensor is its responsivity, or rate of change of optical signal as a function of analyte concentration A good rule of thumb for chemosensor design is that the target association constant should be approximately the inverse of the median guest concentration for the concentration range of interest (Eq 3) Assuming that the amount of guest in solution far exceeds the amount of host in the sensor, this ensures that the concentration of free host is comparable to that of the complex (Equation 4, derived from Eqs 2 and 3)

Thus, fluctuation of guest concentration in either direction from the median will cause significant changes in both the concentrations of [HG] and [H] This approach is best if it is not known whether the optical signal of host or complex will be most responsive to complexation, but there are exceptions For example,

a chemosensor with a much smaller Ka than the inverse of the median guest concentration can be used if [HG] can be detected against a small background

Bell and Hext

signal, as in the formation of a fluorescent complex from a nonemissive chemosensor Measurement of complexation by UV-visible absorption and fluorescence spectroscopy has been treated in greater mathematical detail elsewhere in the literature (Schneider and Yatsimirsky, 2000)

Another issue of great importance in chemosensor design is the choice of chromophore or fluorophore used to report analyte binding In particular, the absorption wavelength must be compatible with the light-absorbing properties of the medium in which measurements are to be made and with the light source For example, proteins absorb ultraviolet light, so optical chemosensors for analytes in biological fluids (e.g., blood) should have ~.max values larger than ca

350 nm Fortunately, Stokes' Law ensures that fluorescent chemosensors will emit light (~em) at longer wavelengths than that used for excitation (~,ex), but practical considerations come into play here, too For reasons of instrument configuration, sensor cost, and light scattering, it may be better to use optical filters to reduce excitation light reaching the detector, rather than to arrange the detector perpendicular to the incident light beam, as in conventional spectrofluorometers Therefore, a large Stokes' shift is generally desired (e.g.,

~em = ~ex > 50 nnl)

Keeping the issues of target analyte affinity and acceptable fluorophore or chromophore wavelengths in mind, the chemosensor designer should proceed to consider binding selectivity, optical signaling mechanism, and the method to be used for immobilizing or delivering the chemosensor These considerations are discussed in the following sections

1.2 Molecular recognition

A chemosensor must recognize its target analyte, much in the manner of picking out a familiar face in a crowd More importantly, it must respond quickly and specifically (e.g., Hi there, Fred!) This selection process can result from selective binding or selective response, but in the latter case interfering substances will competitively inhibit optical response to the desired analyte Having said this, complete specificity for a single potential guest is not necessary because the chemosensor needs to pick out its guest only from the substances typically present in the analyte solution For example, a chemosensor for measuring sodium or potassium in blood need not discriminate against transition metals, unless the test is intended for patients who are already deceased!

How can the molecular structure of a chemosensor be engineered to specifically bind the target analyte? Factors affecting molecular recognition in designed host-guest complexes have been the subject of intense scrutiny over the last three decades (Cram and Cram, 1978; Cram, 1988; Lehn, 1988, 1990; Rebek, 1988, 1990; Schneider, 1991) While there is no reliable way to predict host-guest

selectivity, research in this field and in the broader area of supramolecular chemistry (V/3gtle, 1991; Lehn, 1995; Steed and Atwood, 2000) has identified several intermolecular forces that play important roles The fundamental electrostatic (ion-ion, ion-dipole and dipole-dipole), hydrogen bonding, and van der Waals interactions are of course important, but more subtle forces have also been examined, including n-stacking (Hunter and Sanders, 1990; Hunter, 1993), cation-n interaction (Ma and Dougherty, 1997), CH-rc interaction (Laatikainen et al., 1995; Cloninger and Whitlock, 1998) and solvophobic effects

How can these intermolecular forces be marshaled and controlled to effect molecular recognition? Cram put this task most succinctly in two simple terms: complementarity and preorganization The principle of complementarity is that:

"to complex, hosts must have binding sites which cooperatively contact and attract binding sites of guests without generating strong nonbonded repulsions" (Cram, 1988) Clearly, the number and type of binding sites in the host must match those in the guest to produce an optimally stable complex This principle alone, however, is insufficient for the design of a host that must select between similar guests, such as alkali metal cations differing only in size Here we need guidance from the principle of preorganization: "the more highly hosts and guests are organized for binding and low solvation prior to their complexation, the more stable will be their complexes" (Cram, 1986) Excellent selectivities can be achieved in the alkali metal series with highly preorganized hosts, justifying the description of preorganization as the "central determinant of binding power" (Cram, 1986; Reinhoudt, 1988)

While host preorganization leads to stronger and more selective guest binding, it also increases rigidity In chemosensors, rigidity can hinder access of the analyte

to the binding site, slowing equilibration (Eq 1) considerably This can produce unacceptable delays in equilibrium measurements, simultaneously retarding kinetic measurements, as well The rate of complex dissociation (kout) is the factor limiting equilibration time, but remember that the binding rate (k~) is proportional according to:

Even when Ka is large, kt~ may be too small for kinetic measurements to be made within the 1-2 minute time frame required in certain sensor applications Therefore, chemosensor rigidity must be balanced with flexibility Preorganization does not need to be completely sacrificed, as evidenced by the various degrees of complex stabilization attending the chelate, cryptate and macrocyclic effects (Hancock and Martell, 1988) Indeed, a certain degree of chemosensor flexibility may be desired in order to produce an optical response

by an "induced fit" mechanism, which leads us to the next consideration in chemosensor design

Bell and Hext

Figure 2 Cartoon showing different optical responses of intrinsic and extrinsic

chromophores or fluorophores in chemosensor

1.3 Optical signaling

We have considered the desirable binding and optical characteristics of chemosensors What about the mechanism coupling the binding event with signal transduction via the chromophore or fluorophore? It makes sense that the binding site and the optical reporter should be structurally integrated as much as possible in order to maximize this communication In this context, it is useful to draw a distinction between intrinsic and extrinsic fluorophores or chromophores (Bell et al., 1993; Lakowicz, 1999), as shown in Figure 2

Intrinsic optical reporters are structurally integrated with the analyte binding site

to maximize the influence of the bound guest on the optical properties of the chemosensor Here, chemosensors have a profound advantage over biosensors

It is much easier to build a chromophore or fluorophore into a chemosensor binding site during its synthesis than to modify or introduce an optical reporter into the active site of an enzyme or the recognition site of an antibody Such modifications of biological molecules usually damage their molecular recognition capabilities, so the optical reporter must be conjugated extrinsically

to their binding sites During chemosensor synthesis, optical and other properties

(e.g., pKa) of the chromophore or fluorophore can also be fine-tuned in order to optimize performance

Whether the optical reporter is intrinsic or extrinsic to the molecular recognition site, the molecular mechanism for optical response should be considered during chemosensor design While the mechanism of many known sensors, especially fluorescent chemosensors and biosensors, may not be well understood, Table 1 lists many mechanisms that have been identified and incorporated into chemosensor design Here an important distinction is made between guest binding effects on chromophores vs fluorophores Useful absorbance effects generally result from changes in molecular structure, including proton transfer, other chemical reactions, and isomerization Fluorescence is much more sensitive to subtle changes in the geometry and electronic structure of the ground state, as well as the electronic excited state It is uniquely responsive to physical processes affecting depopulation of the emissive excited state (Lakowicz, 1999), such as conformational restriction occurring upon analyte complexation (McFarland and Finney, 2001; Mello and Finney, 2001) As indicated in Table

1, fluorescent chemosensors can utilize several photophysical processes, in addition to all of the structural mechanisms available to chromophoric chemosensors

The structural changes listed in Table 1 for chromophore signaling generally change the polarity or degree of electronic delocalization (conjugation) within the host chromophore T h e chromophore protonation state can change when a neutral host ionizes during binding of a cationic guest, or when binding drives proton transfer between host and guest or within the host (tautomerization) Guest binding can also change electron distribution in the ground state of the chromophore and the energy of the locally excited (LE) state The resulting

"polarization" mechanism (Table 1) operates in a manner that is similar to solvatochromism (Reichardt, 1979) The LE states of most chromophores are more polar than their ground states, so polar solvents stabilize them more than the ground state The resulting decrease in the energy difference between ground and excited states causes the )~max to shift to longer wavelength as solvent polarity increases (positive solvatochromic effect) A polar guest can, in principle, cause

a bathochromic shift in the absorption of a chemosensor by the same mechanism,

or a specific charge or dipole interaction could cause the opposite effect, a hypsochromic shift

Fluorescence wavelengths are much more sensitive to solvent polarity and are subject to other effects, as illustrated in Figure 3 (Lakowicz, 1999) Solvent (and host-guest) interactions do not have time to adjust immediately to photon absorption and concomitant electronic excitation, occurring on the time scale of

10 ~5 seconds Specific interactions and general solvent relaxation involving partial orientation of solvent dipoles stabilize the polar excited state Relaxation

Bell and Hext

Table 1 Optical Response Mechanisms

Internal charge transfer (ICT)

Twisted internal charge transfer (TICT)

Resonance energy transfer (RET)

Photoinduced electron transfer (PET)

Chromophore

Fluorophore

of polar solvents has a larger stabilizing effect on the excited state than solvation

of the LE state, producing larger bathochromic shifts of fluorescence emission bands Clearly, guest binding can strongly influence the energy of the emissive state of a chemosensor, either by displacing solvent or by introducing new electrostatic interactions in the complex Moreover, many fluorophores can form

an internal charge transfer (ICT) state, also shown in Figure 3, involving transfer

of electron density from electron,donating to electron-accepting groups Interactions of the solvent or the bound guest molecule with these groups will determine the energy of the ICT state and also determine which state has the lowest energy

The twisted intramolecular charge transfer (TICT) state (Rettig, 1994) provides even greater opportunities as a fluorescence sensing mechanism in chemosensors Formation of the TICT state involves rotation of donor and acceptor groups of the fluorophore As stated earlier (Section 1.1), a large difference between absorption and fluorescence wavelengths is desirable in chemosensor design, and TICT emission can produce Stokes' shifts in excess of 100 nm Because formation of the TICT state requires conformational mobility, rigidification of the chemosensor upon guest complexation can profoundly influence the intensity

of long-wavelength TICT fluorescence

Resonance energy transfer (RET), involving through-space jumping of electronic excitation energy from a donor fluorophore to an acceptor fluorophore or a quencher, has been described as "the most general and valuable phenomenon for

Figure 3 Jabtofiski diagram showing effects of specific solvent-fluorophore interactions, general solvent relaxation and formation of internal charge transfer (ICT) states (adapted from Lakowicz, 1999)

fluorescence sensing" (Lakowicz, 1999) Because RET operates over macromolecular distances, it can be used to detect major conformational changes

of nucleotides or association between biomolecules, as in immunoassays RET is not sensitive to changes in donor-acceptor separation in the subnanometer range,

so it has not been found to be useful for detecting changes in the conformation of synthetic chemosensors upon guest binding On the other hand, photoinduced electron transfer (PET) is a very useful sensing mechanism in fluorescent chemosensors (Bissell et al., 1993; Czarnik, 1993a,b, 1994; Desvergne and Czarnik, 1997; Granda-Vald6s et al., 2000) PET quenching of fluorescence occurs when an electron-rich group, such as an amino or phenoxide group, donates an electron to the fluorophore excited state PET is different from ICT in that the electron donor group is not in direct conjugation with the rr system of the fluorophore Binding of an electron-deficient guest to the donor group increases fluorescence emission by stabilizing and decreasing the mobility of donor electrons It is also possible that some chemosensors may operate by PET quenching involving electron transfer from the guest to the fluorophore upon complexation

Bell and Hext

probes Indeed, fluorescent probes of the intracellular concentrations of metals and other analytes have proven to be extremely useful in biomedical research (Lakowicz, 1999) Also, when used to fabricate disposable or reusable sensing materials for sensing instruments, chemosensors may not need to be covalently attached to a surface or other substrate Often they can be hydrophobically adsorbed to a nonpolar surface layer or dissolved in the plasticizer of a polymer film or membrane

Covalent immobilization of chemosensors on surfaces or in materials is often used to improve sensor stability and avoid migration of the chemosensor into the analyte solution Immobilization is usually a late-stage activity in sensor development, but the convenience of adding functional groups or side chains as covalent linkage sites should be considered while planning chemosensor structure and synthesis Incorporation of a tether that does not interfere with binding or optical response can also be used to apply the techniques of polymer supported synthesis and combinatorial chemistry that are now crucial to pharmaceutical development It has been pointed out that both drug discovery and chemosensor development are host-guest research and that bead-based screening of combinatorial libraries should be easily accomplished with fluorescent chemosensors (Czarnik and Yoon, 1999)

2 History

As artificial molecular recognition systems, chemosensors have their roots in coordination chemistry of metals, the lock-and-key model for enzyme action, and the biomolecular receptor These concepts were introduced by Alfred Werner in

1893, Emil Fischer in 1894, and Paul Ehrlich in 1904, respectively Studies in the first half of the 20 th century on hydrogen bonds, clathrates, inclusion compounds, and rr donor-acceptor complexes set the stage for the appearance in the second half of the century of the discipline alternately termed host-guest chemistry (Cram and Cram, 1978; Cram, 1988), supramolecular chemistry (Lehn, 1988, 1990, 1995; V6gtle, 1991; Steed and Atwood, 2000), or molecular recognition (Rebek, 1988, 1990) Macrocyclic ligands for transition metals (Hancock and Martell, 1988) played a special role in this drama, and organic chemists began to take notice in 1967 when Charles Pedersen discovered crown ethers capable of mimicking the alkali metal transport properties of ionophore antibiotics (Pedersen, 1984; Gokel, 1991; Bradshaw et al., 1996; Bradshaw and Izatt, 1997) The design and synthesis of host compounds was recognized as an established field of research by the award of the 1987 Nobel prize in chemistry to Pedersen, Cram and Lehn

Earlier studies in the field of supramolecular chemistry revolved around the complexation of metal cations Incorporation of dyes and fluorophores into the structures of crown ethers gave chemosensors for alkali metal and alkaline earth

metal ions (Takagi and Ueno, 1984; Lbhr and V6gtle, 1985) At about the same time, chelation-based fluorescent probes for intracellular calcium and other metals were developed (Tsien, 1993) Chromogenic reagents for alkali metals based on cryptands, cryptaspherands, and spherands have also been devised (Helgeson et al., 1989; Chapoteau et al., 1993; Dolman et al., 1996) Such reagents are of practical utility in the determination of sodium, potassium, and calcium in blood (Kumar et al., 1988; Chapoteau et al., 1993) More recently, there has been much interest in fluorescent chemosensors for transition metals, mainly involving polyamine ligands (Fabbrizzi et al., 1997a, 1998; Bargossi et al., 2000; Prodi et al., 2000)

Supramolecular chemistry of neutral molecules and anions (Schreeder et al., 1996; Bianchi et al., 1997; Schmidtchen and Berger, 1997) has been explored more recently, and here is where many current challenges for chemosensors lie Many organic analytes of interest in biological systems exist as neutral molecules

or anions (e.g., carboxylates and phosphates) Such relatively complex guest molecules present problems in the design of both the molecular recognition and optical response functions of the chemosensor Therefore, the examples in the following section of this chapter are drawn from recent work on artificial receptors and chemosensors for neutral organic molecules, as well as organic and inorganic anions

3 State of the A r t - Chemosensors for Organic Analytes

The examples of chemosensors in the following section show that a wide range

of analytes can be detected by this approach In selecting these examples, we have used the Czarnik (1994) definition of a chemosensor consisting of a molecule incorporating a binding site, a chromophore or fluorophore, and a mechanism for communicating between the two This excludes reagent approaches involving irreversible formation of colored or fluorescent products (Davis et al., 1999; Lewis et al., 2000) Also excluded by this definition are receptor-based sensing strategies in which the analyte competes for binding and displaces a fluorophore from the receptor, though elegant work has been done in this area (Lavigne and Anslyn, 2001; Wiskur and Anslyn, 2001; Springsteen and Wang, 2001; Cabell et al., 2001)

A key aspect of chemosensor architecture is the attachment or conjugation of a reporter chromophore or fluorophore to a synthetic molecule that is responsible for recognizing the analyte Recently, there have been important advances in developing sensors for organic analytes based on molecularly imprinted polymers (Subrahmanyanet al., 2000; Appleton and Gibson, 2000), but these materials do not fit our chemosensor concept Effective sensing materials (optodes) that are not molecular devices can also be prepared by incorporating ionophores and ionizable chromophores or fluorophores in permeable polymers

Bell and Hext

(Bakker et al., 1997; Murkovic and Wolfbeis, 1997; Spichiger-Keller, 1997; Krause et al., 1999) Finally, cyclodextrins have been used extensively as the recognition component of fluorescent sensor molecules (Ueno, 1993; de Jong et al., 2000; Wang and Ueno, 2000; Narita et al., 2001), but these examples are excluded because cyclodextrins are natural, rather than designed or artifical receptors

As described in the previous section, chemosensors for metal cations have been studied for many years and have been extensively reviewed (Takagi and Ueno, 1984; L/3hr and V/Sgtle, 1985; Czarnik, 1993a,b; Bissel et al., 1993; Fabbrizzi and Pogi, 1995; Dolman et al., 1996; Desvergne and Czamik, 1997; de Silva et al., 1997; Kimura and Koike, 1998a; Fabbrizzi et al., 1998b, 2000; Granda-Vald6s et al., 2000; Yamauchi and Hayashita, 2000; Prodi et al., 2000; Baragossi et al., 2000) While innovative work on chemosensors for metals continues (e.g., Hayashita et al., 2000; Bronson et al., 2001; Baxter, 2001; Raker and Glass, 2001; McFarland and Finney, 2001; Mello and Finney, 2001), the relative maturity of this field of research can be seen from the use of chromogenic chemosensors in clinical chemistry (e.g., Kumar et al., 1988; Chapoteau et al., 1993) and application of fluorescent chemosensors as intracellular probes for metals (e.g., Tsien, 1993; Zalewski et al., 1994; Lakowicz, 1999)

The following examples have been selected from the recent literature to illustrate the chemosensor approach to detecting neutral and anionic organic analytes, as well as inorganic anions Analytes of biological interest, such as carbohydrates, phosphates and blood metabolites, are highlighted In most cases, performance characteristics including sensitivity, selectivity, and absorption or emission wavelengths must be improved to enable practical application Nevertheless, these challenges can be met by tuning chemosensor structure by means of the power of organic synthesis

3.1 Chemosensors for carbohydrates

Development of chemosensors for biologically important carbohydrates has become a target for many research groups over the last decade In general, there have been two approaches to the problem The first approach is based on a rapid and reversible covalent bond formation process (James et al., 1996a,b; Shinkai and Takeuchi, 1996; Shinkai et al., 2000) The second approach utilizes hydrogen-bonding interactions to recognize the carbohydrate (Davis and Wareham, 1999)

Monoboronic acids covalently interact with saccharides in aqueous solutions by formation of the corresponding boronate ester, as shown in Figure 4 with ethylene glycol representing the 1,2-diol unit of saccharides With phenylboronic acid, this only occurs under basic conditions because esterification is favored when the hydroxyboronate anion is produced However, if the aromatic ring is

Figure 4 Reversible reaction of phenylboronic acid with 1,2-diols

made sufficiently electron-deficient (lowering the pKa of the boronic acid), hydroxyboronate anion formation can occur at neutral pH Alternatively, an

acid-base, boron-nitrogen interaction which significantly lowers the pKa of the boronic acid (Wulff, 1982)

The order of affinity of arylboronic acids for monosaccharides is D-fructose > D- arabinose > D-mannose > D-glucose However, for many biological applications

a greater degree of selectivity and sensitivity is required, as well as a different specificity O n e way this can be obtained is by employing suitably designed diboronic acids, with the sugar bridging the space between the two boronic acid groups This has led to receptors that can sense glucose, for example, with good selectivity and sensitivity Two glucose chemosensors that have been developed

by the groups of Shinkai and Norrild are given as examples, as they are considered by these authors to be significant advances in the field

Anthracene receptors 1 (James et al., 1994) and 2 (Eggert et al., 1999) both give optical responses on complexing monosaccharides at neutral pH (Figure 5) In unbound receptor 1, a PET process quenches the fluorescence of the anthracene moiety by electron transfer from the amino group to the anthracene excited state When boronic acids form cyclic boronate esters, the Lewis acidity of the boronic acid is enhanced (Lorand and Edwards, 1959) Thus for receptor 1, monosaccharide binding increases the boron-nitrogen interaction, resulting in suppression of the PET process, leading to enhanced fluorescence For receptor

2, fluorescence of the anthracene moiety is enhanced by saccharide binding, but the reason is unclear Receptor 1 affords greater increase in fluorescence (7-fold

Bell and Hext

v s 2-fold) and greater selectivity for binding of glucose over other monosaccharides, when compared to receptor 2 At pH 7.77 (33.3 % methanol buffer), the stability constants for 1 are: D-glucose (logKa = 3.6); D-allose (logKa

= 2.8); D-fructose (logKa - 2.2); D-galactose (logK~ - 2.2) At pH 7.4 the only stability constant reported for 2 is for D-glucose (logK~ = 3.4) However, receptor

2 has the advantage of being water soluble, which is required for many applications In fact, it has been predicted that a receptor similar to 2 could be used to construct a blood glucose sensor As shown in Figure 5, the proposed structures of the complexes of these glucose receptors involve the pyranose form for 1 (James et al., 1994) and the furanose form for 2 (Eggert et al., 1999) Norrild has proposed that the optical responses of both receptors involve binding glucose in the furanose form (Bielecki et al., 1999), and there is additional evidence to support this case (Cooper and James, 1998)

A "sugar tweezer" that was designed from a boronic-acid-appended ~x- oxobis[porphinatoiron(III)] (3, Figure 6) was shown to have high selectivity, as well as the highest known association constant for glucose (Takeuchi et al., 1996a, 1998) Unfortunately, the absorption spectrum of 3 is not significantly affected by saccharide addition, so an optical response would have to be introduced to enable its use as a sensor The binding of saccharides was

Figure 6 Boronic acid receptors selective for D-glucose (3) and for D-lactulose (4)

observed by circular dichroism spectroscopy, a technique that would be difficult

to adapt to a sensor Also, it would be necessary to lower the pK, of the boronic acid groups in 3 so studies could be performed at physiological pH

Alteration of the distance between two boronic acid groups has also led to receptors for small saccharides (James et al., 1997), as well as di- and trisaccharides An example of a receptor which selectivity binds D-lactulose in methanol is tetraarylporphyrin 4, also shown in Figure 6 (Kijima et al., 1998) Receptors which discriminate between the two enantiomers of a sugar have also been synthesized, though overall the selectivity between two different sugars is not great (Takeuchi et al., 1997a; Mizuno et al., 1999, 2000)

Most carbohydrate receptors based on the hydrogen-bonding approach have been studied by either NMR spectroscopy or circular dichroism (Davis and Wareham, 1999) As sugar binding was apparently not amenable to study by either UV- visible or fluorescence spectroscopy, these receptors will not be discussed in this chapter However, in the case of four receptors, two of which are shown in Figure 7, the binding of carbohydrates was monitored by UV-visible spectroscopy (Rusin and Kr~d, 1999; Kr~il et al., 2001), and for one of these (receptor 5) fluorescence spectroscopy was used as well These receptors show good affinity for some saccharides, even in polar solvents, though overall the selectivity is poor In DMSO (containing 5% methanol) as measured by fluorescence spectroscopy, some association constants for 5 are: o-fructose (log/<, = 1.9); D-a-lactose (logKa- 3.4); D-trehalose (logKa = 3.1); D-maltotriose (logK, = 2.8)

Bell and Hext

Figure 7 Hydrogen-bonding receptors for disaccharides (S) and trisaccharides (6)

However, Rusin and Knil (1999) reported a slight variance in the same association constants as measured by UV-visible spectroscopy Also, the selectivity in water (5 % methanol) was different for D-fructose (logKa = 2.3) and D-o~-lactose (logKa = 2.4) In water (containing 5% methanol), some association constants for 6 are: D-glucose (logKa = 3.1); D-lactose (logKa = 4.5); D- maltotriose (logKa = 4.7) Overall, receptor 6 shows some selectivity for the trisaccharide maltotriose, relative to monosaccharides

There have been a few reports of receptors designed for sensing carbohydrate- like molecules (Takeuchi et al., 1996b, 1997b; Yamamoto et al., 1998) An interesting example is receptor 7 (Figure 8), designed for sensing D-glucosamine hydrochloride (Cooper and James, 1997) The system acts like an AND logic gate in that the anthracene fluorophore is turned off by both amino groups due to PET quenching For the molecule to become fluorescent, two events are required: ammonium binding by the azacrown ether and diol binding by the boronic acid This can be achieved in the binding of D-glucosamine but not of D- glucose

3.2 Chemosensors for other neutral organic molecules

Chemosensors for neutral organic molecules other than carbohydrates can be roughly divided into two types" a molecule that reversibly reacts with a particular functional group or one whereby the receptor has been tailored to selectively detect a specific compound

An approach to the detection of alcohols (Mohr and Spichiger-Keller, 1997; Mohr et al., 1998a) and amines (Mohr et al., 1998b) is based on their reversible reaction with an electron deficient carbonyl functionality that is part of a dye

Figure 9 A reversible reaction-based sensor for alcohols and amines

molecule, as shown in Figure 9 The dye molecule is incorporated into a lipophilic membrane which is then exposed to aqueous substrate solutions Using dye 8 for the sensing 'of alcohols, a catalyst is required to shorten the response time (unlike amines where it was not necessary), and it was found that the response varied with the lipophilicity of the alcohol Formation of the hemiacetal (Sa) is monitored by a hypsochromic shift in the UV-visible absorption spectrum and a decrease in the fluorescence emission spectrum of 8 The sensitivity of a membrane incorporating 8 is lower than that of enzymatic sensors and can not be used in clinical applications However, it is suitable for monitoring ethanol content in alcoholic beverages Membrane incorporation of a perylene-based dye that can react reversibly with aldehydes and ketones has also been used for the optical sensing of aqueous propanal at low pH (Mohr et al., 2000)