Báo cáo khoa học: Electrochemical biosensing with nanoparticles Arben Merkoci pot

MINIREVIEW Electrochemical biosensing with nanoparticles Arben Merkoc¸i Institut Catala ` de Nanotecnologia and Universitat Auto ` noma de Barcelona, Spain Introduction Electrochemical sensing (ES) techniques are playing a growing part in various fields in which an accurate, low cost, fast and online measuring system is required. With regard to quality and cost, ES is better than not only standard analytical methods ⁄ assays but also sen- sors based on other transducing mechanisms. Beside the relatively low cost compared with optical instru- mentation, advantages such as the possibility of minia- turization as well as in-field applications make ES devices very attractive in several fields such as environ- mental monitoring, food quality control and clinical analysis. The ES field, as also mentioned in a recent review by Bakker & Qin [1], is relatively mature and has found its way into commercial products and advanced integrated sensing systems. The use of ES in DNA and immunoanalysis is well known. Electrochemical affinity biosensors based on DNA hybridizations [2] or immunoreactions are playing a more and more important role in DNA and protein analysis. Several ES methodologies for DNA and proteins based on either label-free or the use of enzymes as labels have been reported [1]. The recent developments of bottom-up nanotechno- logy approaches are offering novel materials such as nanoparticles (NPs) with special interest for (bio)analy- sis. This minireview will focus on current progress in applying NPs to DNA sequence determination as well as immunosensing systems based on electrochemical schemes. It first covers some aspects related to electro- chemical properties of metal NPs and then their appli- cations as labels in a variety of DNA electrochemical detection schemes. Electrochemical properties of metal NPs Research on metal and semiconductor NPs [Group II– VI compound semiconductors such as CdSe, ZnSe, CdTe, etc. called also quantum dots, (QDs)] [3] as well as gold NPs (AuNPs) has increased rapidly in recent Keywords conductometric techniques; DNA analysis; differential pulse voltammetry; electrochemical analysis; gold nanoparticles; labelling technologies; nanotechnology; protein analysis; stripping voltammetry Correspondence A. Merkoc¸i, Institut Catala ` de Nanotecnologia, Campus UAB, 08193 Bellaterra, Barcelona, Catalonia, Spain E-mail: arben.merkoci.icn@uab.es (Received 26 September, accepted 8 November 2006) doi:10.1111/j.1742-4658.2006.05603.x This minireview looks at the latest trends in the use of nanoparticles (NPs) in electrochemical biosensing systems. It includes electrochemical characteri- zation of NPs for use as labels in affinity biosensors and other applications. DNA analysis involving NPs is one of the most important topics of current research in bionanotechnology. The advantages of the use of NPs in designing novel electrochemical sensors for DNA analysis are reviewed. Electrochemical NPs can also be used in designing immunoassays, offering the possibility of easy, low cost and simultaneous detection of several pro- teins. Research into NP applications in electrochemical analysis is in its infancy. Several aspects related to sensitivity as well integration of all the assay steps into a single one need to be improved. Abbreviations CV, cyclic voltammetry; DPV, differential pulse voltammetry; ES, electrochemical sensing; HBsAb, hepatitis B surface antibody; HBsAg, hepatitis B surface antigen; MPC, monolayer-protected cluster; QD, quantum dot. 310 FEBS Journal 274 (2007) 310–316 ª 2006 The Author Journal compilation ª 2006 FEBS years because of interest in size-dependent and shape- dependent tailoring of their physical and chemical properties and their potential in applications in catalysis, sensors, and molecular electronics. Finally, research on the electrochemical properties of NPs has received special attention with regard to applications in the electrochemical biosensor field. Quin et al. [4] have studied differential pulse voltam- metry (DPV) responses of thiolate monolayer-protec- ted Au clusters (Au 147 MPCs). They showed 15 evenly spaced (DV) peaks characteristic of charge injection into the metal core (Fig. 1, left). This was clear confir- mation that MPCs behave as multivalent redox spe- cies, in which the number of observable charge states is limited by the size of the available potential window. The electrochemistry of QD-size Au MPCs has also been studied by Murray and coworkers [5]. According to these authors, Au MPCs behave as multivalent redox species as charge injection into the core is quant- ized [6]. Using the ‘particle in a box’ model, Brus [7] predic- ted the dependence of redox potential on particle size for CdS QDs. However, this model has not been tested by electrochemical measurements of QDs in solution, largely because of the limited solvent window of many solvent ⁄ electrolyte systems and the instability of the particles. Haram et al. [8] have reported the novel use of elec- trochemistry of CdS QDs in N,N¢-dimethylformamide. They were able to show a direct correlation between the electrochemical band gap and the electronic spectra of CdS NPs in N,N¢-dimethylformamide. In the light of this and the irreversibility of oxidation and reduc- tion of Q-CdS, the authors propose a multielectron transfer process in which the electrons are consumed by fast-coupled chemical reactions through decomposi- tion of the cluster. Essentially, the electron is scav- enged immediately after injection into the particle, and, unlike the case of thiol-capped metal particles, the CdS QDs can accept additional electrons at the same potential, giving rise to higher peak currents. The appearance of additional cathodic and anodic peaks in the middle of the potential window supports this. A typical cyclic voltammetry (CV) for thioglycol-capped CdS Q-particles at the Pt electrode is given in Fig. 1 (right) where clear oxidation and reduction peaks are apparent at )2.15 V (A1) and 0.80 V (C1), respect- ively. This is compared with the response of the sup- porting electrolyte alone. DNA analysis The development of sensitive nonisotopic detection systems has significantly affected the DNA sensor field. Affinity electrochemical biosensors based on enzyme labeling solved the problems of radioactive detection (e.g. health hazards and short lifetimes) and opened up new possibilities in ultrasensitive and automated bio- logical assays. Nevertheless, biological research and other application fields need a broader range of more 12 0 1- 2- 0.0 1. 0 2.0 V2.1 ERQsusr e v V /E/E -I / nA 0.1 0.0 0. 1 - 0 . 2 - 0. 3 - E H N su s r e v V 0 . 2- 0.0 0.2 Current (µA) SdC-Q etylortceleesaB 1C 2C 3C 3A 2A 1 A Fig. 1. Left, DPV responses for MPC solutions measured at a Pt microelectrode; as-prepared 177 lM C6S-Au 147 (upper) showing 15 high- resolution QDL peaks, and 170 l M C6S-Au 38 (lower) showing a HOMO-LUMO gap. It can be seen that the as-prepared solution contains a residual fraction of Au 38 which smears out the charging response in E regions where quatized double layer charging (QDL) peaks overlap. The electrode potential scanned negative to positive. The caption single electron-transfer events are termed quantized double layer charging. Adapted from [4]. Right, CV response in the absence and presence of thioglycol-capped CdS Q-particles (1 mgÆmL )1 fraction IV) at a Pt electrode. Sweep rate, 50 mVÆs )1 and tetrahexylammonium perchlorate (THAP), 0.05 M. Adapted from Fig. 1 of reference [8]. A. Merkoc¸i Electrochemical biosensing with nanoparticles FEBS Journal 274 (2007) 310–316 ª 2006 The Author Journal compilation ª 2006 FEBS 311 reliable, more robust labels to enable high-throughput bioanalysis and simultaneous determination of mul- tiple-molecule types present in a sample [9]. The DNA-recognition event can be detected using different strategies, including intrinsic electroactivity of the nucleic acid [10], DNA duplex intercalators [13], electroactive markers [14], enzyme labels [15], etc. The existing labeling techniques have several drawbacks; the markers used have short life times and a limited number of combinations that can be practically used for simultaneous analysis of various analytes. Fluorescent labeling of biological materials with small organic dyes is also widely used in the life sci- ences and has been used in a variety of DNA-sensing systems based on optical detection. Organic fluoroph- ores, however, have characteristics that limit their effectiveness for such applications. These limitations include narrow excitation bands and broad emission bands with red spectral tails, which can make simulta- neous evaluation of several light-emitting probes problematic because of spectral overlap. Also, many organic dyes exhibit low resistance to photodegrada- tion. To improve assay sensitivity and achieve better and more reliable analysis, there is a great demand for labels with higher specific activity. The electrochemical properties of NPs make them extremely easy to be detected with simple instrumenta- tion. Sensitivity, long life time, and multiplexing capa- bility have led to the explosive growth of NP-based DNA electrochemical assays in recent years [11,12,16– 18]. NPs are made of a series of semiconductor NPs which are easily detected by highly sensitive techniques such as stripping methods among others. In addition, these electrochemical properties may allow simple and inexpensive electrochemical systems to be designed for detection of ultrasensitive, multiplexed assays. Conductometric techniques Detection of DNA hybridization used in connection with conductometric measurements after labeling with AuNPs has been successfully demonstrated by Mirkin’s group [19]. They exploited the silver-deposition technique to construct a sensor based on conductivity measurements. A small array of microelectrodes with gaps (20 lm) between the electrodes leads is construc- ted. DNA probe sequences are then immobilized on the substrate between the gaps. By using a three-component sandwich approach, hybridized target DNA is used to recruit AuNP-tagged reporter probes between the electrode leads. The NP labels are then developed in the silver enhancer solution leading to a sharp fall in the resistance of the circuit (Fig. 2A). Electrochemical stripping Other electrochemical techniques for DNA detection have been reported. Voltammetric or potentiometric stripping analysis using mercury film electrode depos- ited on a pencil graphite or glassy carbon electrode has been used. The intrinsic electrochemical signals of AuNPs, observed after dissolving these with HBr ⁄ Br2, are then related to DNA. This is achieved by pre-con- centration of gold(III) ions through electrochemical reduction and subsequent determination by anodic- stripping voltammetry [20] (Fig. 2B). Going further to lower the detection limits, gold tra- cer ‘amplification’ by silver deposition on the surface has also been applied [21,22]. This is a clever way of achieving higher sensitivities for DNA detection (Fig. 2C). The labeling of probes bearing different DNA sequences with different NPs enables the simultaneous detection of more than one DNA target in a sample. The number of targets that can be readily detected simultaneously (without the use of high level multi- plexing) is controlled by the number of voltammetri- cally distinguishable NP markers. A multitarget sandwich hybridization assay involving a dual hybrid- ization event, with probes linked to three tagged inor- ganic crystals and to magnetic beads has been reported [23]. The DNA-connected QDs yielded well-defined and resolved stripping peaks at )1.12 V (Zn), )0.68 V (Cd) and )0.53 V (Pb) at the mercury-coated glassy carbon electrode (versus the Ag ⁄ AgCl reference elec- trode) after acidic dissolution of the above metal NPs (Fig. 2D). Single nucleotide polymorphisms were also detected recently by Liu et al. [24]. They used ZnS, CdS, PbS, and CuS NPs linked (using phosphoramidite chemistry through a cysteamine linker) to adenosine, cytidine, guanosine, and thymidine mononucleotides, respect- ively. They introduced the monobase-conjugated nano- crystals to the hybrid-coated magnetic-bead solution. Each mutation thus captures different nanocrystal- mononucleotides and in this way the unknown single nucleotide polymorphisms were detected on the basis of distinct voltammetric stripping signals. Making the DNA ⁄ NP detection system more integrated The use of NPs as electrochemical labels for DNA sens- ing has several advantages. The related technique – stripping voltammetry – is cheaper, faster and easier to use in field analysis than optical ones. Moreover it offers the possibility of simultaneous detection of Electrochemical biosensing with nanoparticles A. Merkoc¸i 312 FEBS Journal 274 (2007) 310–316 ª 2006 The Author Journal compilation ª 2006 FEBS several biological molecules in the same sample using a unique sensor because of the distinct voltammetric waves produced by different electrochemical tracers. The advantages offered along with the possibility of being used in several biosensing systems based on electrochemical techniques require the development of novel NP-detection strategies that avoid dissolution of NPs before detection thereby integrating the whole assay. A novel NP-based system for detection of DNA hybridization based on magnetically induced direct electrochemical detection of a 1.4-nm Au67 QD tag linked to the target DNA has been reported by our group [25]. The Au67 NP tag is directly detected after the DNA hybridization event, without the need for acidic (i.e. HBr ⁄ Br 2 ) dissolution. In this way, the NP- detection event is integrated into the biosensor system. The binding of a DNA probe to paramagnetic beads was achieved by streptavidin–biotin interaction. The resulting DNA-modified paramagnetic beads were then hybridized with the DNA target labeled with Au67 NP in a 1 : 1 ratio. The resulting Au67-DNA probe–DNA target paramagnetic bead conjugate was collected mag- netically on the surface of a transducer with built-in magnet (Fig. 3). The two main highlights of this novel genosensor are that: (a) the direct voltammetric detec- tion of metal QDs obviates the need for their chemical dissolution; (b) the Au67 QD–DNA probe ⁄ DNA tar- get-paramagnetic bead conjugate does not create the interconnected 3D network of Au-DNA duplex–para- magnetic beads as in previously developed NP DNA assays. In this way, the sensitivity of the assay is not decreased by the sharing of one gold tag by several DNA strands, achieving lower detection limits. The magnetically triggered Au67 NP direct detection meth- odology described above can be applied to different bioassays (including isoelectric immunoassay). Current efforts in our laboratory are aimed at broadening the application range of the QD direct detection protocol and the development of a microfluid device to integ- rate all the steps of the genomagnetic protocol on a lab-on-a-chip platform. The integration of nanotechnology with biology and electrochemistry is going to produce major advances in dC +2 C d S uC + 2 nZ + 2 A g A u A u 3 + A u e - gA uA gA uA Z n S C u S A B C D PM PM PM gA + Fig. 2. Strategies used for DNA detection by labeling with metallic NPs. Usually a probe DNA has been immobilised on a transducing plat- form and then hybridized with target DNA and further with NP-modified DNA probes. (A) Conductivity assay in which gold is accumulated in the gap and later on a silver enhancement procedure in the presence of hydroquinone is performed. (B) Electrochemical stripping assays based on labeling with AuNPs which were then dissolved with HBr ⁄ Br 2 and detected by stripping techniques. (C) The same as (B) but the AuNPs are first covered with silver by a deposition treatment and then detected by stripping techniques via a silver enhanced signal. (D) Multilabelling by the use of three different NPs (QDs) and the simultaneous detection of the three DNA targets. A. Merkoc¸i Electrochemical biosensing with nanoparticles FEBS Journal 274 (2007) 310–316 ª 2006 The Author Journal compilation ª 2006 FEBS 313 the field of electrochemical DNA sensors. Research on the application of these systems to real samples is in progress and is expected to produce novel alternatives for DNA analysis. Protein analysis Electrochemical immunosensors, based on the coupling of immunochemical reactions with electrochemical transduction, have attracted considerable interest in recent years. The use of NPs as either immobilization platforms or labels in immunosensing systems has been reported. NPs as protein immobilization platforms The combination of self-assembly with NPs has attrac- ted researchers to develop novel immunosensors. A hepatitis B surface antigen (HBsAg) immunosensor has been developed by self-assembling AuNPs to a thiol-containing sol–gel network. The hepatitis B sur- face antibody (HBsAb) was adsorbed to the surface of the AuNPs and used later to detect HBsAg in human serum based on the specific reaction of HBsAb with HBsAg. The electrochemistry of the ferricyanide redox reaction was used as a marker to probe the interface and as a redox probe to determine HBsAg [26]. Self-assembly to immobilize HBsAb on a platinum disk electrode based on AuNPs, Nafion, and gelatin as matrices has also been demonstrated. Detection is based on the change in the electric potential before and after the antigen–antibody reaction. The proposed hepatitis B immunosensor provides a novel tool for directly monitoring the concentration of HBsAg in serum samples [27]. NPs as labels for proteins The study of immunoreactions through labeling with metallic ions after a long period of using only enzymes or dyes opened up new possibilities. The interaction of human serum albumin with its antibody was used as a model system, with bismuth ion serving as the metal label that is detected by potentiometric stripping analysis [28]. The concept of this single-use stripping immunosensor opened up the way to improving the AB CDE Fig. 3. Schematic representation of the analytical protocol (not in scale). (A) Introduction of streptavidin-coated paramagnetic beads; (B) immobilization of the biotinylated probe (DNA2) on the paramagnetic beads; (C) addition of the 1 : 1 Au67–DNA1 target; (D) accumulation of Au67–DNA1 ⁄ DNA2–paramagnetic bead conjugate on the surface of the magnetic electrode; (E) magnetically triggered direct DPV electro- chemical detection of Au QD tag in Au67–DNA1 ⁄ DNA2–paramagnetic bead conjugate. Also shown is the schematic of the integrated geno- sensor based on labeling with Au67 NPs. The magnetic field produced by a tiny magnet introduced inside a graphite epoxy composite electrode attracts the resulting Au67 QD–DNA hybrid–paramagnetic bead conjugate. In situ electrochemical oxidation of the AuNPs followed by differential pulse voltammetry of the gold ions is then performed and the signal obtained related to the quantity of the DNA target found in the sample. Adapted from reference [25]. Electrochemical biosensing with nanoparticles A. Merkoc¸i 314 FEBS Journal 274 (2007) 310–316 ª 2006 The Author Journal compilation ª 2006 FEBS performance of immunoassays by incorporating electro- chemical NPs as labels. NP-based electrochemical biosensors for disease- related glycan markers based on their interaction with surface-functionalized lectins have been developed [29]. The assay has been optimized and tested using a model system. It involves immobilization of the lectin, the carbohydrate-recognition element, on the gold surface and the following competition between a nanocrystal (CdS)-labeled sugar and the target sugar for the carbo- hydrate-binding sites on lectins. Finally, the extent of competition is monitored by highly sensitive electrochemical stripping detection of the captured nanocrystal. An electrical immunoassay-coding protocol for the simultaneous measurement of multiple proteins based on the use of different inorganic nanocrystal tracers has been developed. The concept is demonstrated for a simultaneous immunoassay of b 2 -microglobulin, IgG, BSA, and C-reactive protein in connection with ZnS, CdS, PbS, and CuS colloidal crystals, respectively [30]. The assay shows the efficient coupling of the multipro- tein electrical detection with the amplification feature of electrochemical stripping transduction yielding fem- tamolar detection limits. In addition, the proposed method is combined with efficient magnetic separation so as to minimize nonspecific binding effects. NPs as aptamer labels Aptamers offer great promise for sensitive displace- ment assays, as the tagged protein has a significantly lower affinity for the aptamer than for the unmodified analyte. The use of nanocrystal tracers for designing multianalyte electrochemical aptamer biosensors with subpicomolar (attomolar) detection limits has been demonstrated. A simple single-step displacement assay was used. Several thiolated aptamers were first immo- bilized on the gold substrate. The corresponding QD-tagged proteins were then bound and this was followed by the addition of the protein sample. The displacement was monitored through electrochemical detection of the remaining nanocrystals (PbS and CdS). The concept has been demonstrated for dual- analyte sensing (thrombin and lysozyme) and could easily be expanded for the simultaneous measurement of a large panel of proteins [31]. Conclusions The integration of nanotechnology with biology and electrochemistry is expected to produce major advan- ces in the field of electrochemical biosensors. Recent progress has led to the possibility of the application of electroactive NPs to simple and low cost analytical sys- tems for analysis of biological molecules, such as pro- teins and nucleic acids. NPs show great promise for electrobioanalytical applications. They can be used as labels for affinity biosensors, biomolecule immobilization platforms, and biocatalysts in various bioassays. The electrochemical detection of NP labels in affinity biosensors using stripping methods allows the detailed study of DNA hybridization as well as immunoreac- tions, with possible genosensor and immunosensor applications. The use of diverse NPs for the simulta- neous detection of several biomolecules is expected to open up new opportunities for DNA diagnostics. The developed electrochemical coding is being adapted to other multianalyte biological assays, particularly immu- noassays. The electrochemical coding technology is thus expected to open up new opportunities not only for DNA diagnostics but for bioanalysis in general. NPs have a promising future in the design of electro- chemical sensors, but their use will be driven by the need for smaller detection platforms with lower limits of detection. The use of electrocatalytic NPs, already demonstrated in the ultrasensitive micro RNA assay [32], can be exten- ded to several other applications. 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