Biosensors and Bioelectronics 21 (2006) 1424–1433

Review

State of the art and recent advances in immunoanalytical systems

Christophe A. Marquette ∗ , Loı̈c J. Blum
Laboratoire de Génie Enzymatique et Biomoléculaire, UMR 5013, CNRS Université Claude Bernard Lyon 1, Bât. CPE,
43 Bd. 11 Nov. 1918, 69622 Villeurbanne, Cedex, France
Received 11 June 2004; received in revised form 2 September 2004; accepted 3 September 2004
Available online 6 December 2005

Abstract
This article is an overview the state of the art and the recent developments in immunosensors. Homogeneous immunosensors, heterogeneous
immunosensors, integrated immunosensors and biochip format immunosensors are presented, based on optical, electrochemical, magnetic or
mechanical detection/transduction systems.
© 2005 Elsevier B.V. All rights reserved.

Keywords: Electrochemical; Immuno-chip; Immunosensor; Magnetic; Mechanical; Optical

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1424
2. Generalities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1425
3. Homogeneous phase immunosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1425
4. Heterogeneous phase immunosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1426
4.1. Based on chromatography techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1426
4.1.1. Electrochemical detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1426
4.1.2. Optical detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1427
4.2. Integrated systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1427
4.2.1. Electrochemical detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1428
4.2.2. Quartz crystal microbalance (QCM) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1428
4.2.3. Optical detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1428
4.2.4. Magneto-immunosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1430
4.2.5. Micromechanical cantilever-based immunosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1430
5. The immuno-chip . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1431
6. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1431
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1432

1. Introduction use and safety, since radiolabels and radio-labelled molecules

are restricting in term of handling, storage and elimination.
In the last 10 years, immuno-enzymatic methods (ELISA: Concomitantly to the arising of those classical ELISA tests
“Enzyme Linked Immunosorbent Assay”) acquired more and on microtiter plates, a large number of immunosensor systems
more popularity to the detriment of the radio-immunoassay were described in the literature. Based on the biosensor technolo-
(RIA). The main reasons of this growing interest are ease of gies, those sensors attempted to bypass the inherent problems
of the microtiter plate tests and particularly the time consump-
tion. Most of the developed immunosensors include a sensing
∗ Corresponding author. Tel.: +33 472 44 82 14; fax: +33 472 44 79 70. layer supporting a particular immobilised antigen or antibody.
E-mail address: [Link]@[Link] (C.A. Marquette). The solid support used is generally in close contact with a trans-

0956-5663/$ – see front matter © 2005 Elsevier B.V. All rights reserved.
doi:10.1016/[Link].2004.09.037
 C.A. Marquette, L.J. Blum / Biosensors and Bioelectronics 21 (2006) 1424–1433 1425

Fig. 2. Schematic representation of the competitive and sandwich type immuno-

tests.

Fig. 1. General representation of the immunosensor functional architecture. rarely use incubation times long enough to reach the steady
state.
In steady state conditions, the amount of labelled antibodies
ducer needed for the detection of the formed immune complex (Ab* ) (or antigens), linked to the immobilised antigens (Ag) (or
(Fig. 1). antibodies) depends only on the affinity constant K (Eq. (1)).
The strategy is, then, on the one hand to use the high sensitiv- Conversely, when the steady state could not be reached because
ity of the detection/transduction systems, in order to minimize of time or diffusion limitations, the amount of immobilised
the time measurement, and on the other hand to take advantage antigens will have a great influence on the performances of
of the regeneration of the immobilisation support to perform the test.
various analysis with a single immunosensor. Most of the competitive immunosensors could, then, be
The detection systems involved are directly related to the considered as analytical systems, which quantify the amount
labelling, enzymatic or not, performed on the antigen or on the of “still-free” labelled antibodies after a definite incubation
antibody. For each particular detection type, a specific labelling time with the antigen of the sample (Mallat et al., 2001). In
will be preferred, even if some labels could be used with dif- those conditions, the larger the amount of immobilised anti-
ferent detection methods (i.e. the horseradish peroxidase which gens, the faster the reaction will be with the antibodies to be
could be used for fluorescence, chemiluminescence, absorbance measured and the better the performances of the immunosen-
and electrochemical measurements). In the present review, the sor.
different immunosensors are grouped in categories according to Those considerations are useful to understand why immo-
the detection method used. bilisation supports with high specific surface such as porous
surfaces, macrometrics membranes (with micron size thick-
2. Generalities ness), and polymeric gels are usually preferred:
ka ka [Ab∗ : Ag]
Theoretically, all immunochemical techniques could be Ab∗ + Ag
Ab∗ : Ag, K= = (1)
used to design immunosensors. Nevertheless, because the kd kd [Ab∗ ][Ag]
immunosensors are offered as rapid testing systems, the reac-
tion conditions, and particularly the reagent concentrations (in 3. Homogeneous phase immunosensors
competitive format) and the incubation times have to be adapted.
Most of the developed immunosensors are based either on Most of the immunosensors described in the literature are
competitive or sandwich assay, when applied to the detection based on the separation of the antigen:antibody complexes by
of low (herbicides, toxins) and high (proteins, cells) molecular immobilising one or the other reagent. Nevertheless, homoge-
weight molecules, respectively (Fig. 2). neous phase immunosensors were proposed, which could detect
Two approaches could be considered when dealing with the formation of the immune complex without separation. Those
competitive immunosensors. A first-one in which immobilised systems are based on the physico-chemical properties of lumi-
antibodies react with free antigens in competition with labelled nescent molecules.
antigens. A second-one, using immobilised antigens and labelled The light emission of a luminescent molecule such as isolu-
antibodies, is generally preferred and prevents all the problems minol, linked to an antigen was shown to be increased following
related to antibody immobilisation (loss of affinity, orientation the reaction with a specific antibody (Schroeder et al., 1976;
of the immobilised protein). Kohen et al., 1979). Nevertheless, the light emission increasing
A few theoretical aspects are presented herein since, com- level highly depends on the antigen structure and on the inter-
pared to immuno-tests on microtiter plate, immunosensors action types involved during the recognition by the antibody
1426 C.A. Marquette, L.J. Blum / Biosensors and Bioelectronics 21 (2006) 1424–1433

(Weeks, 1992). Those systems could not, then, be considered as

generic tools for the development of homogeneous immunosen-
sor.
Based on the labelling of the antigen with a fluorescent
molecule, few immunosensors were developed for the detec-
tion, via fluorescence quenching, of the antigen:antibody com-
plexes in an homogeneous phase. An example of those sys-
tems is an immunosensor for the detection of the pesticide
2,4-dichlorophenoxyacetic acid (2,4-D) (Matveeva et al., 1997).
Fluorescein was linked to the antigen and a decrease of its fluo- Fig. 3. Principle of the double label-based homogeneous immunosensor. E1 and
E2 are two enzymes working sequentially.
rescence emission intensity was observed during the association
between the labelled antigen and the specific antibody. The pres-
ence of free antigen (un-labelled) from the sample induced, then, 4. Heterogeneous phase immunosensors
an increase of light emission, due to the displacement of the
labelled antigen from the recognition site of the antibody. Detec- 4.1. Based on chromatography techniques
tion limits of 10 ␮g/L could be obtained with this system, but the
experiments with real samples suffer from an extensive purifi- 4.1.1. Electrochemical detection
cation step prior to the measurement. Those systems are composed of a static phase on which the
This point is a general problem encountered with homo- antigens or antibodies are immobilised. This phase is localised
geneous immunosensors. Indeed, since those systems are in a column named immunoreactor (IR), spatially separated
based on low light emission variations, generated by physico- from the detection system and connected to this latter via a
chemical environment modifications of the luminescent reagent flow (Fig. 4A). The supporting phase could be made
molecules, the presence of excessive concentration of pro- up of silanised porous glass beads (Controlled Pore Glass:
teins or other high molecular weight structures have to be CPG, 240–200 ␮m in diameter) (Botchkareva et al., 2002),
avoided. pre-activated commercial Eupergit beads (150 ␮m de diamètre)
The use of an electrochemiluminescent system appeared to (Wilmer et al., 1997), glass (Jiang et al., 1995) or quartz capillary
be an interesting way to deal with these problems. (Trau et al., 1997) modified with PEG (polyethylene glycol) or
Indeed, a recently published study presents the use of poly-l-lysine, respectively. A few studies were, also, reported
electrogenerated luminol chemiluminescence in homogeneous using membrane type static phase (Krämer and Schmid, 1991)
immunosensor (Qi and Zhang, 2004). Digoxin was labelled with packed in the immunoreactor.
luminol through a luminol–BSA–digoxin conjugate. The for- Table 2 summarised the label enzyme/substrate couples
mation of the immunocomplex induced a loss of the luminol typically used in the electrochemical detection of the anti-
electrochemiluminescent signal. When use in a competitive for- gen:antibody complex. In the particular case of the sensor using
mat, free digoxin could be detected as low as 0.3 ␮g/L and an immunoreactor, the reaction products are carried to the detec-
satisfactory results were obtained when applied to human con- tion cell via the reagents flow. In a similar way, this flow enables:
trol serums. (i) the delivery of the immunochemical reagents (antigen or anti-
Another homogeneous immunosensor format, dedicated to body), to the static phase, (ii) the achievement of incubation
high molecular weight molecules, was presented a few years while stopping or decreasing the flow rate (Trau et al., 1997)
ago (Ngo and Lenhoff, 1981; Sevier et al., 1981). It is based and (iii) the wash out of the un-reacted species by flowing buffer
on a sequence of two enzymes, used as labelled for two mon- through the immunoreactor.
oclonal antibodies (Fig. 3). Each antibody is produced against Successful immunodetection of pesticides such as 2,4-D and
a specific epitope of the target molecule. When both antibodies atrazine was reported using the present immunoreactor system
are linked to the target, the enzymes are close enough to induce (Wilmer et al., 1997; Jiang et al., 1995; Trau et al., 1997; Vianello
an increase of the reaction sequence rate. Table 1 presents the et al., 1998). Detection limits of 0.1 ␮g/L were obtained, with
different enzymes’ systems, which could be used in those devel- either immobilised antibodies or antigens, and with acceptable
opments. incubation times, from 3 to 20 min. Only two of the mentioned

Table 1
Detection systems for double labelling homogeneous immunosensors (from Sevier et al., 1981)a
Systems Labels (L1 /L2 ) Substrates Products (P1 /P2 )

Confine enzymes Hexokinase/G6PDH ATP/glucose NAD+ Glucose-6-phosphate/gluconolactone-6-phosphate + NADH + H+

Confine enzymes GOD/HRP Glucose/HRP substrate H2 O2 /HRP substrate
Bioluminescent system NAD; FMN oxidoreductase/bacterial NADH/FMN FMNH2 /hν
luciferase
Enzyme/substrate HRP/luminol H2 O2 hν
a L1 and L2 are the labels for each monoclonal antibody; P1 the primary product (common intermediate) and P2 is the final product. G6PDH, glucose-6-phosphate
deshydrogenase; GOD, glucose oxidase; HRP, peroxidase; H-donor, hydrogen donor; hν, emitted light.
 C.A. Marquette, L.J. Blum / Biosensors and Bioelectronics 21 (2006) 1424–1433 1427

Fig. 4. Schematic representation of the immunoreactor based immunosensor using: (A) electrochemical detection or (B) optical detection. P: pumping system, I:
injection system and IR: immunoreactor.

studies (Wilmer et al., 1997; Trau et al., 1997) proposed the In a similar way, alkaline phosphatase widely used
regeneration of the immobilised material by flowing a chaotropic for electrochemical detections, could be used as optical
solution (Glycine 0.1 M/HCl pH 2.5) through the immunore- label in conjunction with a chemiluminescent substrate:
actor. The immunosensor could, then, be re-used 20–30 times CSPD (disodium 3-(4-methoxyspiro[1,2-dioxetane-3,2 -(5 -
without loss of sensitivity. chloro)tricyclo[3.3.1]decan]-4-yl)phenyl phosphate) (Dzogoev
et al., 1996).
4.1.2. Optical detection In both cases, the emitted photons could be detected with
Optical immunosensors using an immunoreactor are based light measurement devices such as a photomultiplier tube or
on the same principle as electrochemical immunosensors except a charge coupled device camera (CCD). Those highly sensi-
that the detection system must be in close contact with the reac- tive and low-noise systems usually enable the achievement of
tor, particularly in the case of a chemiluminescent detection sensitive immunosensors. Moreover, since chemiluminescent
(Fig. 4B). When a fluorescent detection is used, the detector reactions do not suffer from the classical interferences problems
is usually positioned sequentially to the immunoreactor. The associated to the electrochemical measurements, those optical
different immobilisation supports and the label enzymes used immunosensors could be used directly with complex samples
for the previous electrochemical immunosensor type could be without pre-treatment or purification.
used for the optical systems. Nevertheless, the reactions involved
during the detection will be different. 4.2. Integrated systems
Horseradish peroxidase could be used for chemilumines-
cent (Gonzalez-Martinez et al., 2001; Ramanathan et al., 2002; Integrated immunosensors are based on the spatial integra-
Botchkareva et al., 2002) or fluorescent detection (Krämer and tion of the sensing layer and the detection/transduction system
Schmid, 1991; Gonzalez-Martinez et al., 1997) in conjunction (Fig. 5). The immobilisation of the immunochemical reagents
with a fluorogen substrate (example: hydroxyphenyl propionic could, then, be performed on a support, bring in close contact
acid: HPPA). The detection limits obtained with those systems with the transducer, or directly on the transducer. This last con-
for pesticides (atrazine, carbaryl, irgarol 1051) are usually below figuration with a sensing layer localised at the surface of the
0.1 ␮g/L with analysis times of 20 min. One of those systems transducer allows new detection methods. Thus, piezo-electric
shows a high stability which enables a 600 times regeneration of and magneto sensors, sensors using evanescent field or surface
the immunoreactor with negligible loss of sensitivity (Gonzalez- plasmon resonance (SPR), involve direct immobilisation meth-
Martinez et al., 2001). ods on the transducer.

Table 2
Enzyme labels used in electrochemical immunosensor developments
Enzyme Substrates Potential (electrode) References

Alkaline phosphatase p-Aminophenyl phosphate +150 mV (carbon) Trau et al. (1997)

Alkaline phosphatase Phenyl phosphate +870 mV (carbon) Wehmeyer et al. (1986) and Doyle et al. (1984)
Alkaline phosphatase p-Hydroquinone phosphate +300 mV (carbon) Vianello et al. (1998)
Glucose oxidase Glucose/O2 +650 mV (platinum) Tsuji et al. (1990) and Carter et al. (1993)
Glucose oxidase Glucose/benzoquinone +400 mV (carbon) Weetall and Hotaling (1987)
Catalase Hydrogen peroxide Oxygen electrode Aizawa et al. (1980)
Peroxidase Hydroquinone/hydrogen peroxide −300 mV (gold) Skládal and Kalab (1995)
1428 C.A. Marquette, L.J. Blum / Biosensors and Bioelectronics 21 (2006) 1424–1433

limit of 6 × 103 cells/mL could be obtained for the detection of

Escherichia coli with a sandwich type immunosensor based on
this principle (Ruan et al., 2002).
Finally, field effect transistors (FET) and particularly ion
sensitive FETs (ISFET), have to be presented as a basis for
immunosensor developments. The main part of an ISFET is ordi-
nary metal oxide silicon FET (MOSFET) with the gate electrode
replaced by an ion selective membrane, a solution and a refer-
ence electrode. The nature of the membrane/insulator will, then,
give the ion specificity of the sensor (pH, H2 , NH3 ). The pH sen-
Fig. 5. General representation of a flow injection integrated immunosensor. P: sitive IFSETs are the most widely used sensor for immunosensor
pumping system, I: injection system and SL: sensing layer. developments with a large range of possible insulators (SiO2 ,
Si3 N4 , Al2 O3 and Ta2 O5 (Yuqing et al., 2003)) and enzyme
labels (urease, peroxidase and glucose oxidase).
4.2.1. Electrochemical detection FET are usually presented as robust devices which could be
Integrated electrochemical immunosensors, when based on stored without particular conditioning and presenting a good
label reagents, make use of the same label enzymes as the response to pH variations in a wide range of temperature. Nev-
immunoreactor-based immunosensor (Table 1). Nevertheless, ertheless, only a few examples of ISFET-based immunosensors
because immobilisation supports and detection systems are in could be found in the literature (Tsuruta et al., 1995; Starodub et
contact or merging, the electroactive reaction products will be al., 2000; Selvanayagam et al., 2002; Plekhanova et al., 2003).
detected concomitantly to their production (Ghindilis et al., This lack of experimental systems could be related to on the
1998; Bakker, 2004). one hand some difficulties encountered in the immobilisation of
Numerous systems were described in the literature using immuno-reagents at the FET surface and on the other hand to a
immobilisation support as different as carbon electrodes (Rosen relatively low ion specificity and a complicated microelectronic
and Rishpon, 1989; Dzantiev et al., 1996; Kröger et al., 1998; technology.
O’Regan et al., 2002), gold electrode (Rosen and Rishpon,
1989), different membrane types (Immobilion® , Carter et al., 4.2.2. Quartz crystal microbalance (QCM)
1993; Carter et al., 1994, cellulose, Aizawa et al., 1980) or Quartz crystal immunosensors are based on the immobili-
electrogenerated polytyramine film (Tsuji et al., 1990). The per- sation of antigen or antibody at the surface of a piezo-electric
formances of the corresponding analytical systems are usually material. The intrinsic vibration frequency of the support is,
identical to those obtained with immunoreactor-based systems then, modulated, following the immunochemical recognition
(Section 4.1.1). Nevertheless, integrated approaches permit to reaction. Such analytical systems usually suffer from interfer-
consider a potential miniaturisation of the sensor. ences problems when used in complex matrices. Those problems
Other electrochemical systems, based on classical labels are related to temperature and conductivity variations of liquid
(alkaline phosphatase, peroxidase) enable the measurement media, which can modify the frequency measurements (Mallat
without detection of the reaction product. Those measurements et al., 2001). Nevertheless, a few recent studies, particularly for
are based on the variations of electron transfer to an electroactive the detection of living cells, show good performances with detec-
compound contained in the solution (Fig. 6). The enzyme label tion limits of Salmonella enteritidis of 104 and 105 cells/mL (Si
is, then, used to generate a precipitating product, which induces et al., 2001; He et al., 2001; Wong et al., 2002).
variations of the electron transfer through the layer immobilised This immunosensor type, using immobilised antibodies,
on the electrode (Katz et al., 2001; Ruan et al., 2002). The could, also, be applied to the detection of compounds in gaseous
transducer could be a gold or indium tin oxide (ITO) electrode samples (Stubbs et al., 2002). Thus, numerous tests were
modified with thiol or silane function, respectively. Detection described for the detection of the pesticide parathion and its
derivatives (methyl parathion, malathion) (Ngeh-Ngwainbi et
al., 1986). The response time of the sensor was, then, as fast as
1–2 min with a detection limit of 36 ppb.
Interesting studies on QCM sensors were focused on the
amplification of the signal measured via an amplification of the
mass increasing detected at the surface of the sensor. This was
achieved by labelling the immuno-reagents with large entities
such as latex beads (Aizawa et al., 2003) or gold nanoparticles
(Ma et al., 2002).

4.2.3. Optical detection

[Link]. Fluorescence. Fluorescent immunosensors could be
Fig. 6. Example of precipitating substrate-based electrochemical immunosen- divided into two main groups: (i) based on the separation of
sor. HRP: horseradish peroxidase. the antigen:antibody complex, before the measurement and (ii)
 C.A. Marquette, L.J. Blum / Biosensors and Bioelectronics 21 (2006) 1424–1433 1429

Fig. 8. Principle of the evanescent wave-based immunosensor. EW: evanescent

wave and n1 and n2 : core and external medium refractive indexes, respectively.

immunosensors (Section 4.1.2). Antibodies or antigens are then

immobilised either at the surface of a macrometric membrane
(Marquette et al., 1999), bring into contact with a fibre optic,
Fig. 7. Schematic representation of a flow injection integrated fluorescent or directly at the end of this fibre (Marks et al., 2000). This
immunosensor. Em: emitted light, Ex: excitation source, SL: sensing layer and latter enables the transfer of the photons, produced during the
FO: fibre optic. catalysed reaction, to a light measurement system (usually a
photomultiplier tube).
using the transducer properties to distinguish between the pro- For particular application, the immunosensor could be inte-
duced immuno-complex and the free labelled molecules. grated in a continuous flow device, which enables the semi-
The first-ones meet with the concept of integrated systems, automation of the system (Fig. 9A) (Marquette et al., 1999;
with, most of the time, antibodies or antigens immobilised on Marquette and Blum, 2000). Such flow immunosensors were
a solid support in close contact or directly on the surface of successfully applied to the detection of trace level of plank-
an optical fibre ending in a continuous flow cell (Fig. 7). The tonic toxin in seafood (okadaic acid) and pesticide (2,4-D), with
fibre is, then, used as a light guide for both the excitation and detection limits of 0.1 ␮g/L and assay times of 20 min.
the emission light. The reagents flow enables the carrying of Non-enzymatic electrochemiluminescent labels were, also,
the reacting species to the sensing layer and the excess reagent described in the development of immunosensors. Under elec-
washing. Detection limits of the order of ␮g/L were obtained trochemical oxidation, these molecules, luminol (Marquette and
with such systems for the detection of explosives (Bakaltcheva Blum, 1999) or ruthenium complexes (Michel et al., 1999; Yoon
et al., 1999) and pesticides (Xing et al., 2000). et al., 2003), generate excited species, which emit a photon
In the second configuration, the sensor uses a particular
property of the wave guide. Indeed, while an electromagnetic
wave travels, in particular conditions, through a wave guide, an
evanescent wave appeared at the core/external media interface
(Fig. 8). This wave could be used to excite fluorescent molecules
in the wave penetrating zone, i.e. a few hundreds nanometers.
The labelled antigens, which have reacted with the antibodies
immobilised at the wave guide surface could, then, be detected
without separation of the antigen:antibody complexes. Numer-
ous studies have shown the wide possibilities of such a technique,
particularly in the field of pesticides detection (Mosiello et al.,
1997; Wittmann et al., 1996; Oroszlan et al., 1993; King et al.,
2000). The immunochemical reaction could, also, be monitored
continuously, leading to a real time measurement of the forma-
tion of the immuno complex.

[Link]. Chemiluminescence and electrochemiluminescence.

Numerous immunosensors based on chemiluminescent detec- Fig. 9. Schematic representation of: (A) a flow injection integrated chemilu-
tion were described. Those sensors involve the label enzymes minescent and (B) electro-chemiluminescent immunosensor. SL: sensing layer,
presented above for the development of immunoreactor-based FO: fibre optic, FC: flow cell and GCE: glassy carbon electrode.
1430 C.A. Marquette, L.J. Blum / Biosensors and Bioelectronics 21 (2006) 1424–1433

The detection limits obtained with these systems compared well

with those obtained with the systems described above and using
labelling of the immuno complex. Response times were, also,
acceptable and could be lowered to less than 1 min in particular
cases (Severs et al., 1993).
A new improvement of the technique was recently proposed
by using a Gold Binding Protein (GBP) in order to enhance the
density of immobilised antibodies on gold surface (Soh et al.,
2003).

4.2.4. Magneto-immunosensors
Magneto-immunosensors are based on either the labelling of
immuno reagents with magnetic particles or the use of magnetic
beads as a solid phase.
In the first case, the label particles enable the enhancement
of the detected signal obtained from a piezo-electric sensor. The
presence of the magnetic particle increases the mass difference
Fig. 10. Principle of the surface plasmons resonance (SPR)-based immunosen- generated after the immuno recognition (Kim et al., 2003). Thus,
sor. Change of the SPR angle (θSPR) following the immunochemical reaction. a sandwich assay was designed for the detection of the pathogen
Salmonella typhimurium. The detection limit was lowered from
during its relaxation to a stable energy level. A direct immobil- 105 to 102 cells/mL by using the magneto enhancement.
isation of the antigen at the surface of a glassy carbon electrode The second analytical use of the magnetic beads has been
was, then, used to enable the electro-catalysis of the lumines- widely studied in conjunction with different detection methods.
cent reaction (Fig. 9B). The performances obtained for the 2,4-D Usually, antibodies or antigens were immobilised at the surface
detection were similar to those obtained with peroxidase labelled of magnetic beads and incubated in homogeneous conditions
antibodies (Marquette and Blum, 1999). with the corresponding immuno reagent. A magnetic separation
An interesting concept was, also, developed by using mag- took, then, place to enable the washing of un-reacted species and
netic beads as immobilisation phase in conjunction with the detection of the formed immuno complex. An early demon-
peroxidase-labelled antibodies (Wilson et al., 2003). In the stration of the technique was presented by Gehring et al. (1996),
present system, one of the chemiluminescent reaction sub- based on the electrochemical detection of alkaline phosphatase
strate, i.e. the hydrogen peroxide, is generated via oxygen elec- label. The magnetic beads bearing immuno complex were, then,
trochemical reduction at gold electrode. The immuno-beads localised at the surface of graphite electrodes, which formed
were magnetically located at the surface of the electrode and the bottom of a 96 wells plate. More recently, fluorescent and
the pseudo electrochemiluminescent signal recorded. Detection electroluminescent assay format were developed using similar
limits of 0.11 and 19.8 ppb of TNT (trinitrotoluene) and PENT approaches (Yu et al., 2000; Yuan et al., 2003; Tanaka et al.,
(pentaerythritol tetranitrate), two explosive compounds, were 2004). Finally, the easy magnetic beads localisation was used to
obtained. generate a sensing layer at the surface of a piezo-electric sensor
(Li et al., 2003). The magnetic beads bearing antibodies were,
[Link]. Surface plasmon resonance (SPR). Surface plasmon then, immobilised with the help of a permanent magnet at the
resonance is based on the generation of an electromagnetic field surface of a quartz microbalance.
at the surface of a metallic conductive material, under the excita- A third application of magnetic beads is the labelling of
tion with an incident light beam having defined wavelength and antibody or antigen for the magneto-detection of the immune
angle. Excited electrons are, then, generated at the surface of the complex. The detection is, then, based on the perturbation of a
metal and are producing oscillations. The energy absorption dur- magnetic field, which could be quantified using a suitable elec-
ing the resonance induces, then, at a particular incident angle, a tronic device (Richardson et al., 2001; Enpuku et al., 2001).
minimum light reflection. This particular angle (θSPR) depends
on the wavelength and the polarisation used but, also, on the 4.2.5. Micromechanical cantilever-based immunosensors
dielectric properties of the local metal environment. This char- The use of micro-cantilever as force sensors in AFM (Atomic
acteristic is used to detect the interactions between antibodies Force Microscopy) is, therefore, a well-established application.
and antigens at the surface of the metal (Fig. 10). The measure- Silicon, silicon oxide and silicon nitride cantilever are com-
ment of the angle deviation is, then, the basis of the detection. mercially available with different shapes, dimension and force
Thus, SPR-based immunosensors, as the systems using evanes- sensitivities. In the last decade, several groups observed that
cent wave or piezo-electric quartz, could performed real time micro-cantilever could transduce a number of different signal
measurements of the antigen:antibody interactions. domains, e.g. mass, temperature, electromagnetic field, stress,
SPR immunosensors were developed for the detection of into a mechanical deformation (Raiteri et al., 2001). In a similar
pathogen agent (syphilis) in blood (Severs et al., 1993) or pesti- way, a change of bending or resonance frequency of the sensor
cides in soil (Skládal et al., 1999; Minunni and Mascini, 1993). could lead to highly sensitive probing of the cantilever surface.
 C.A. Marquette, L.J. Blum / Biosensors and Bioelectronics 21 (2006) 1424–1433 1431

Two main uses of the micro-cantilever are merging in immu- reference electrodes and platinum auxiliary electrodes. Capture
nosensor developments, based on either stress or frequency antibody were immobilised in a plasma-polymerised double
change measurements. layer of methyldisiloxane. At the present time, only two spe-
Stress or deflection detection mode could be achieved via: (i) cific antibodies were immobilised, against ␣-1-fetoprotein and
optical deviation, (ii) optical interference and (iii) capacitance ␤2 -microglobulin. The measurement is based on the electro-
measurement. In immunosensor technology, the first method, chemical detection of hydrogen peroxide produced by the label
based on the detection of the deviation of a light beam focused enzyme: glucose oxidase.
on the sensor, is preferred (Grogan et al., 2002). A single example was found about arraying piezo-sensor
An interesting study was performed by Ruan’s group on the (Wang et al., 2004). Four piezo-sensors were brought together
use of resonance frequency change of micro-cantilever as detec- in a thermostated flow cell and were enabled to work separately.
tion system for antibody:antigen interactions (Ruan et al., 2003). The sensors were used to detect living leukemic blasts cells and,
The sensor, made of a micrometer-scale magnetoelastic can- moreover, to distinguish different phenotypic lineages of cells.
tilever is coated onto one side with anti-E. coli antibodies as The sensors appeared to work properly and exhibited a detection
recognition layer. A sandwich assay is performed with a second limit of 104 cells/mL in an un-purified media.
antibody labelled with alkaline phophatase. This label, used in Finally a new detection system was published recently
conjunction with a precipitating substrate (5-bromo-4-chloro- based on ellipsometry imaging (Bae et al., 2004). Antibod-
3-indolyl phosphate) enables the enhancement of the sensor ies specific for Yersinia enterocolitica were immobilised at a
frequency change, leading to a highly sensitive detection of E. gold surface deposited via a self-assembled monolayer of 11-
coli (102 cells/mL). mercaptoundecanoic acid. Each steps of the proteins immo-
bilisation, i.e. each layers of proteins, could be monitored by
5. The immuno-chip imaging elipsometry and, finally, the presence of the pathogen
was detected with a detection limit of 103 cell/mL. This study
The immuno-chips follow directly the studies performed in represents an interesting approach since no labelling is involved
immunosensor developments but, also, all the technological and that the surface used, the thiol modified gold, is usual in
advances obtained with the DNA chip improvements. Indeed, micro-array technology.
numerous techniques, and particularly the biological molecules
immobilisation procedures could be transferred from DNA chips 6. Conclusion
to immuno-chips. They are, then, used to detect either simulta-
neous compounds in a particular sample or a single compound Despite an interesting concept, immunosensors have not met
simultaneously in different samples. the industrial developments suggested by the abundant litera-
The first immuno-chip developments were carried out, as in ture and the good performances described. One exception is the
the DNA chips field, with fluorescent labels, which still repre- instrumentation based on the surface plasmon resonance (BIA-
sent the majority of the studies (Sapsford et al., 2002; Delehanty Core). Nevertheless, this type of technology is rarely used for
and Ligler, 2002; Melnyk et al., 2002; Avseenko et al., 2002). routine immuno-detection, but finds great application in study-
Interesting results were obtained with an immuno-chip enabling ing the target–receptor interactions. This relative immunosen-
the detection of nine different toxic entities (enterotoxin B, sors failure is generally linked to their format. Indeed, these
ricin, toxin choleric, Bacillus anthracis, Bacillus globigii, Fran- systems were, most of the time, developed as mono-parameter
cisella tularensis, Yersinia pestis, coliphage et S. typhimurium) tests, which could, only, analyse the samples one by one, at
(Taitt et al., 2002). The immobilisation of the bio-molecules was the opposite of the microtiter plate classical approach. Another
performed via a biotin–avidin bridge at a glass slide surface. point is that the regeneration of the sensing layer was during
Unfortunately, the fluorescent detection only permitted qualita- many years a major goal of the studies, leading most of the time
tive measurements. to inaccurate systems, and single use, portable and light devices
This point is a general remark about fluorescent biochips and, were for a long time neglected.
the numerous background and quantitative measurement prob- The immuno-biochips, at the present time under study or
lems have led to the exploitation of well known and characterised development, are not only miniaturising microtiter plate. More
detection/transduction systems already used in immunosensors than the parallelisation of the measurements, already performed
technology. on microtiter plate, the immuno-biochips shows a new func-
Thus, electrochemical (Tang et al., 2002a,b; Skládal and tionality due to the integration of a part or the entire detection
Kalab, 1995) and chemiluminescent (Dzogoev et al., 1996; system. This approach enables, on the one hand a lowering of
Yakovleva et al., 2002) immuno-chips were developed for pes- the operating times of the different test steps and on the other
ticides detection (2,4-D, atrazine). This chemiluminescent chip hand a real integration in a complete system incorporating par-
type is usually considered as more powerful since no complex ticularly microfluidic parts. The latest studies have shown that
signal addressing is required and because a single measurement complete systems integrating samples transport and preparation,
enables the acquisition of the entire chip signals. incubation and washing steps, and detection system could be
An electrochemical immuno-chip was, also, developed based achieved on a few cm2 . These ␮TAS devices (micro total anal-
on a 36 platinum electrodes array (Kojima et al., 2003) integrated ysis) are already under development and represent the future of
in a glass substrate together with a set of thin film Ag/AgCl the immuno-tests.
1432 C.A. Marquette, L.J. Blum / Biosensors and Bioelectronics 21 (2006) 1424–1433

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