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

Electrochemical affinity biosensors based on DNA hybridizations [2] or immunoreactions are playing a more and more important role in DNA and protein analysis.. called also quantum dots, Q

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.

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 (Au147 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, biobio-logical research and other application fields need a broader range of more

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Fig 1 Left, DPV responses for MPC solutions measured at a Pt microelectrode; as-prepared 177 lM C6S-Au147(upper) showing 15 high-resolution QDL peaks, and 170 lM C6S-Au38(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)1fraction IV) at a Pt electrode Sweep rate, 50 mVÆs)1and tetrahexylammonium perchlorate (THAP), 0.05 M Adapted from Fig 1 of reference [8].

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

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⁄ Br2) 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

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C +

n

Z +

Ag

Au

Au 3+

Au

e

-g A u A

g A u A

ZnS

CuS

P M

P M

P M g

A +

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.

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 sursur-face 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

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].

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 b2-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 Future applications in biosensors may aim to reduce the oxidation overpoten-tial in novel electrochemical recognition biosensors

Acknowledgements

This work was financially supported by the Spanish

‘Ramo´n Areces’ foundation (project ‘Bionanosensores’) and MEC (Madrid) (projects MAT2005-03553 and NANOBIOMED, CONSOLIDER)

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