Development of electrochemical immunosensing systems with renewable surfaces

E L S E V I E R

Biosensors & Bioelectronics Vol. 13. No. I, pp. 7-17, 1998 © 1998 Published by Elsevier Science S. A.

All rights reserved. Printed in Great Britain

PII: S0956-5663(97)00096-1 0956-5663/98/$19.00

Development of electrochemical immunosensing systems with

renewable surfaces

Marta Santandreu, Silvia Sold, Esteve Fabregas & Salvador Alegret

Grup de Sensors and Biosensors, Departament de Qufmica, Universitat Autrnoma de Barcelona, 08193 Bellaterra Catalonia, Spain

(Received 3 April 1997; revised version received 22 July 1997; accepted 27 August 1997)

Abstract: The repeated use of immunochemically modified solid phases in ele, ctrochemical immunosensor analysis is the driving interest of this work. Two new strategies have been developed. One of these strategies is aimed at the development of a manual methodology. It comprises the construction of arnperometric immunosensors based on rigid biocomposites. These biocomposites are formed by a conducting polymer composite matrix that acts as a reservoir of an immobilized immunologic material. The surface of the bioc- omposite can be renewed by a simple polishing procedure. The second strategy involves the design of an automatic methodology. It features an immunochemical analytical system using flow injection techniques. The potentiometric detection uses a solid phase formed by immunologic reagents immobilized in magnetic particles. These particles are fixed to the sensor with the use of a magnetic field. The renewal of the reactive surface is achieved by the release and activation of the restraining magnetic field and the manipulation of the flow.

The analytical properties of these immunosensors were evaluated measuring RIgG using a competitive technique and measuring GaRIgG with a sandwich methodology. The labelling enzymes of the immunoconjugates were peroxidase in amperometric measurements and urease in potentiometric measurements. © Published by Elsevier Science Limited

Keywords: immunosensor, renewable immunosensor, immunocomposite, mag- nel:oimmunosensor, flow injection immunoanalysis

INTRODUCTION

The high selectivity displayed between the antibodies and their antigens has motivated the development of immunoassay analytical techniques. These assays are an attractive alternative to classi- cal analytical methods for their sensitivity and simplicity (Marco et al., 1995; Killard et al., 1995). When instrnmental detection methods are followed, the immunologic reaction is usually detected by means of labelled species.

An immunoassay can take many different forms. Heterogeneous techniques, where one of the immunologic reagents is immobilized in a solid phase, are very advantageous (Monroe, 1984; Blake & Gould, 1984). Immunosensors constitute a specific case of the heterogeneous technique. The main novelty of these devices is the integration of the immunologic material to a transducer. This integration increases sensitivity and simplifies the analytical process. However, these sensors have the difficulty of renewal of

Marta Santandreu et al. Biosensors & Bioelectronics

the sensitive surface when they are in continuous use. The need for renewal arises from the high affinity constants derived from the strong antigen- antibody reaction (Marco et al., 1995). This hin- ders their repeated use or the regeneration of the material immobilized in the solid sensing phase.

Two different strategies may be followed to achieve the renewal of the sensing surface:

• breakage of the antibody-antigen bond and reusing the immunologic reagent immobilized in the solid phase;

• elimination of the antigen-antibody complex from the solid support and immobilization of fresh immunologic material.

Several methodologies for the breakage of the antibody-antigen bond are found in the literature. Some of these techniques involve the use of solutions with a low pH and a high ionic strength, such as: 100mM citric acid/HC1; 100mM glycine/HC1 at pH 2; 8 M urea; 6 M guanidine chloride at pH 1.5; 100 mM NaOH and acetonitr- ile, pepsine (2 mg/ml) in 10 mM phosphate at pH 1.9 or the use of proteases to degrade the antibodies (De Alwis & Wilson, 1985, 1987; Robin- son et al., 1986; Ilchman et al., 1990; Lee & Meyerhoff, 1988, 1990; Polzius et al., 1993; Wijesuriya et al., 1994; Tsuji et al., 1990). How- ever, these extreme conditions may denaturalize the immobilized material bringing a radical alter- ation of its identity.

A second alternative is to eliminate the proteic material from the surface. The techniques described in the literature include the deadsorp- tion of the material (Gyss & Bourdillon, 1987) or the breakage of the covalent bond that fixes the antibody to the sensing surface (Boitieux et al., 1987). The electrochemical cleaning of the solid phase has also been reported (Huet & Bour- dillon, 1993).

All these regeneration methodologies try to preserve the recognition capacity of the immobilized immunologic material or the sensing characteristics of the transducer surface.

Two novel strategies for the renewal of immunologic surfaces are presented here. They are applied to two different electrochemical immunosensor systems. These strategies are based on two very different methodologies, one using amperometric immunosensors based on immunocomposites and the other featuring potentiometric magnetoimmunosensors coupled to flow injection systems.

In immunocomposites, one component of the immunologic pair is blended into the bulk of a rigid graphite-polymer conducting composite. This composite acts as a reservoir for the immunologic material. The surface of the sensor is regenerated when it is polished, exposing fresh immunocomposite ready to be used in a new immunoassay. A great variety of enzyme-based biocomposites used in amperometric sensing have been developed in our laboratories (Alegret et al., 1993, 1996a, b; Gal~in-Vidal et al., 1995; Martorell et al., 1994; Crspedes et al., 1993, 1994, 1995; Morales et al., 1996). The development, for the first time, of a rigid immunocomposite used in an amperometric immunosensor has been reported recently by us (Santandreu et al., 1997). That work is revised and widened here in order to augment the sensitivity and the reproducibility of the renewable surface of the immunosensor.

In the case of the magnetoimmunosensor system, the immunologic material is immobilized in the surface of magnetic particles. These particles are transported by a flow system and are retained in close proximity to the potentiometric solid- state sensor by a magnetic field produced by a magnet. This magnetoimmunosensor is part of an automatic flow injection system. The regeneration of the immunologic material is achieved by releasing the magnetic field, thus freeing the particles that are washed by the flow system. New magnetic particles are injected and retained on the surface of the potentiometric sensor by reactivating the magnetic field. A fresh and reproducible surface is thus produced, ready for the next analytical cycle. The same general principle, albeit with different flow systems, has been described previously (Pollema et al., 1992; Stocklein & Schmid, 1990). However, these systems do not have immunosensors as such but immunoreactors that are independent from the sensors, where the magnetic particles are fixed to the inner walls of the main flow channel using a magnet placed upstream from the sensor. In the present reigort, the immunologic phase is on the transducer (immunosensor) and implies a higher sensitivity since the product of the labelling enzyme reaction is not dispersed by the flow system but is measured directly. A new microcell has been designed to accommodate the immunologic reaction, the detection and the magnetic field. The microsensor used is based on a pH- sensitive field-effect transistor (pH ISFET).

Biosensors & Bioelectronics Development of electrochemical immunosensing systems with renewable surfaces

The main features of the two novel strategies for the renewal of immunologic surfaces are presented below. The innovations were evaluated by applying the immunosensors to the measurement of RIgG (rabbit IgG) using a competitive technique. They were used also to detect GaRIgG (goat anti-rabbit IgG) using a sandwich technique. Peroxidase is used as an enzyme label in amperometric measurements and urease in potentiometric measurements.

EXPERIMENTAL

Apparatus

The characterization and evaluation of the amperometric immunosensors used a three-electrode voltammetric system with a platinum auxiliary electrode and a double junction Ag/AgC1 reference electrode (Orion 900200). The reference electrode had 0. l M KC1 as the external reference solution. An LC-.4C Amperometric Controller (BAS Bioanalytical Systems, USA) was used for amperometric measurements. A Labograph E586 recorder (Metrohm, Switzerland) registered the response curve.

Solutions in the flow system are impelled by peristaltic pumps (Minipuls 3, Gilson, France). The 14-way valve is from Rheodyne (California) and the four- and six-way valves were obtained from Omnifit (UK). The sensor was a pH-sensitive field-effect transistor (ISFET) supplied by the Centre Nacional de Microelectr6nica (Barcelona). The reference device was a AglAgC1 double junction electrode (Orion 900200). The signal was measured using an ISFETmetre (Centre Nacional de Microelectr6nica, Barcelona) and recorded with a Labograph E586 chart recorder (Metrohm, Switzerland).

ite powder with a particle size of 50/xm (Merck, Germany), Sealer-Healer 1540 commercial methacrylate (monomer QM-57T-D) from Rohm and Haas (Croydon, UK) and benzoyl peroxide (Fluka, Switzerland) as the hardener.

A dialysis tube (Medicell International, Lon- don, UK) was used to obtain dialysis membranes. These tubes are made of regenerated cellulose which contains glycerin, water and 0.1% sul- phur, approximately.

The magnetic particles were Dynabeads M-280 (2.8/xm diameter) activated with a tosyl group. They were obtained from Dynal (Dynal, Unipath, Barcelona).

Bovine serum albumin (BSA) was obtained from BDH (UK). Hydrogen peroxide, Tris(hydroxymethyl) aminomethane and EDTA from Merck (Germany), and potassium chloride and urea came from Fluka Chemie (Switzerland). Ammonium chloride was purchased from Panreac (Barcelona, Spain).

All other reagents were of the highest grade available. Aqueous solutions were prepared with double distilled water.

Amperometric immunosensors based on immunocomposites

Construction of the amperometric immunosensors Fig. 1 depicts the construction of the immunosensor. The RIgG-methacrylate-graphite composite was prepared by mixing graphite and de- aerated methacrylate monomer in a ratio of 1:1 (w/w). RIgG was then added to obtain a final composition of 0.9% (w/w). This immunocomposite was cured in a nitrogen atmosphere at room temperature for 3 days (Santandreu et al., 1997). The storage temperature for the immunosensors was 278 K.

Reagents

The immunologic reagents used were RIgG (I- 5006), GaRIgG (whole molecule) (R-2004), rabbit anti-goat IgG (whole molecule) peroxidase (HRP) conjugate (RaGlgG-HRP) (A-5420), goat anti- rabbit IgG (whole molecule) peroxidase (HRP) conjugate (A-6667) and goat anti-rabbit IgG (whole molecule) urease conjugate (U-1374). All of these reagents were from Sigma (St Louis, MO).

Immunocomposites were prepared using graph-

Regeneration of the immunosensor surface Before each use, the surface of the electrode was wetted with double distilled water and then polished thoroughly for a few seconds, first with abrasive paper and then with alumina paper (polishing strips 30144-001, Orion). The immunosensor surface was finally cleaned by sonication in double distilled water for 2 min.

Preparation of the incubating solutions A blocking buffer containing 0.1% (w/v) BSA in 0.1 M phosphate buffer at pH 7-0 was used.


B

. m

D

F

Fig. 1. Construction of the amperometric immunosensor. (A) Female connector formed by a 2 mm gold-plated beryllium-copper contact and a polyimide moulding; (B) Bakelite washer; (C) metallic nut; (D) circular copper piece cut from a copper sheet and soldered to the connector end; (E) PVC tubing (6 mm inner diameter, 18 mm

length); (F) final aspect of the assembly with a 3 mm thick cup supporting the immunocomposite.

A GaRIgG standard solution was prepared, dissolving 7.45 mg of GaRIgG in 1 ml of blocking buffer for the sandwich assay. The commercial solution of RaGIgG peroxidase conjugate was diluted with BSA 1% (w/v) prepared in the same phosphate buffer, obtaining a concentration of 0.99% (v/v).

To develop the competitive binding assay, a RIgG standard solution was prepared, dissolving 4.95 mg of RIgG in 1 ml of blocking buffer. The commercial solution of GaRIgG peroxidase conjugate was diluted with BSA 1% (w/v) prepared in the same phosphate buffer, obtaining a concentration of 0.99% (v/v). The washing solution was 0.1 M disodium phosphate, 0.1 M KC1 pH 7.5 buffer.

Competitive binding assay of rabbit IgG Defined volumes of a standard RIgG solution were mixed with a fixed volume of the solution of GaRIgG peroxidase conjugate and sufficient 0.1% (w/v) BSA solution to give a final volume of 90/zl. The mixture was preincubated at room temperature for 30 min while stirring continu- ously. Forty-five microlitres of this solution was placed in the surface of the immunosensor and allowed to stand for 30 rain at room temperature. At the end of this period, the immunosensor was rinsed using the washing buffer.

Sandwich assay of goat anti-rabbit IgG Defined volumes of a standard GaRIgG solution were mixed with enough 0.1% (w/v) BSA solution to give a final volume of 45/zl. This volume was placed on the immunosensor surface and was incubated at room temperature for 30 min. The surface was rinsed with washing buffer. After- wards, a 45/zl drop of the RaGIgG peroxidase conjugate standard solution was placed on the surface of the immunosensor and allowed to stand for 30 rain at room temperature. At the end of this period, the immunosensor was rinsed with the washing buffer.

Amperometric measurements o f peroxidase activity After the immunosensor had been incubated, its surface was covered with a piece of dialysis membrane to enhance the signal-to-noise ratio (Santandreu et al., 1997). The activity of the peroxidase label in the antibody was measured using an amperometric technique with hydroquinone in solution as a mediator to quantify the amount of the antigen-antibody complex on the surface of the sensor. Peroxidase is regenerated after the enzymatic reduction of H202 by transfer- ring electrons through the mediator from the electrode to the enzyme (K~lab & Skl~dal, 1995). A three-electrode voltammetric system was used and

10


a potential of - 0.1 V was applied to monitor the reduction of hydroquinone. A standard solution of peroxide was added to 20 ml of a 0-1 M phosphate, 1-8 x 10 - 3 M hydroquinone and 0,1 M KC1 buffer mixture (pH 7.0) to give a final solution of 4.7 x 1[0 -4M H202, and the steady- state response of the amperometric system was noted.

Magnetoimmunosensor integrated to a flow system

Immobil izat ion procedure A given quantity of magnetic particles (activated with free tosyl groups) was washed with a 0-1 M buffer solution at pH 7.5. RIgG was added after the wash cycle to the particle suspension in the same buffer solution until a concentration of 80 mg/ml was attained. The mixture was left still at 310K, using gentle agitation for 24h. After this time, the residual groups were deactivated and all non-specific adsorptions were blocked by washing three times with PBS/BSA (0.15 M NaC1 in 0.01 M sodium phosphate at pH 7-5 and 1% (w/v) BSA). A further wash with Tris/BSA (0.2M Tris pH 85 with 0.1% (w/v) de BSA) was performed. The particles were then stored in a PBS solution containing BSA 0.1% (w/v) at 277 K for several :months.

Magne to immunosensor The sensor is integrated to the flow system using a methacrylate cell. This cell was made of two blocks measuring 2.5 x 4.0 x 3 cm 3. The cell design is depicted in Fig. 2. The ISFET was fixed in a flat cavity of one of the blocks. The other block had two holes for the entry and exit of the flow. A fle, xible sheet made of silicone rubber was placed between the two blocks for better sealing. This; sheet defined a hollow of a volume of approx!imately 25 ~1 communicating the sensitive area of the ISFET with the entry and exit holes.

The magnetic irnmunosensor was prepared by injecting 50~1 of magnetic RIgG particles (1.5 mg/ml). The peristaltic pump impelled these particles to the sensor where they were restrained by a magnetic field generated by a cylindrical neodymium magnet placed in a cavity situated underneath the sensing area.

C

V

Magnetic particles

ol,

C V B

u F

Fig. 2. Schematic of the cell integrating the magnetoimmunosensor to the flow system. The module is made o f two blocks measuring 2.5 x 4 x 3 cm 3. (A) Machined methacrylate block where an entry channel is perpen- dicular to the sensitive surface while the exit channel is placed at a 45 ° angle; (B) ISFET mounted on a IC board; (C) perforated silicone sheet; (D) flow exit; (E) flow entry; (F) cylindrical magnet placed in a cavity

of the methacrylate bed.

F low system The flow system shown in Fig. 3 comprehends all stages needed for a competitive immunoassay. Solutions of GaRIgG urease conjugate and sample (RIgG) were diluted in a Tris/EDTA solution in BSA 0.1% (w/v) (Glazier & Rechnitz, 1991). These solutions were injected consecutively using the 14-way valve in an air carrier (Alerm & Bartrolf, 1995). They were driven to a preincubation chamber where they remained for 10 min. After this time, 200/~1 of this solution were injected on a 0.1 M NH4CI carrier solution (0.02 ml/min). This carried it to the sensitive gate of the ISFET where the RIgG magnetic particles were fixed. The low flow rate allowed the second incubation to take place here.

After 15min, the flow rate was raised to 0.2 ml/min followed by the injection of 50/xl of enzymatic substrate (0.1 M urea). The pH change induced in the injection zone by the products of the enzyme reaction was detected as a signal peak by the pH-sensitive ISFET.

Fig. 4 shows the reactions taking place in the vicinity of the ISFET gate.

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. . . . . . . . T 3

magnetic particles ~ l ' " ~ "1" ........................................... ~ \ ~ .":)1

R I_&C':_ ~ , ~ . ""-~. magnet

Air

GaRIgG 2

A~r

Fig. 3. Schematic representation of the flow injection immunoassay (FIIA) system. (1) Injection valves; (2) preincubation chamber with magnetic stirring; (3) magnetoimmunosensor.

H2N-CO-N H2

GaRIgG ~ I ~ % ~ v ~ t ~ ~ ~l~ CO 2 + 2 NH 3

Lv ~ RIgG

magnetic particle

............. pH-sensitive ISFET gate

MAGNET

Fig. 4. Schematic representation of the magnetoimmunosensor with the immunologic interactions and the enzyme reactions taking place.

Surface regeneration After recording the signal peak, the magnetic field was released and the flow rate was increased. After the magnetic particles were washed down by the flow system, the magnet was placed again near the sensor and fresh particles were injected.

In this way, new and reproducible surface is present for the following new analytical cycle.

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RESULTS AND DISCUSSION

Amperometric immunosensors based on immunocomposites

Reproducibility o f ,~,he response for different regenerations To optimize chemical regeneration it is necessary that the antigen and the antibody show a low affinity (Huet & Bourdillon, 1993). This is not the case in the immunologic system used in the present work. Some of the methods reported elsewhere and mentioned in the Introduction above use extreme pH values. However, this con- dition may cause the denaturalization of the immobilized antige, n (RIgG), thus affecting its capacity to bind to its corresponding antibody. In the case of the immunocomposites this is not a limitation, since regeneration is achieved by superficial polishing.

Fig. 5 shows the reproducibility of the amperometric response when the surface of an immuno-

-5000

-5500

-60OO

-6500

~-7000

-7500

-8000

-8500

-9000

G, •

i i i i i i r

0 1 2 3 4 5 6 7

Assay number

Fig. 5. Reproducibility of the amperometric response after repeated polishing of the surface of a graphite- methacrylate immunosensor containing 0.9% (w/w) RIgG. Amperometric measurements of peroxidase activity were realized by adding a standard solution of H202 to 20ml of O. I M phosphate, 1.8 × 10 3M hydroquinone and 0.1 M KCI pH 7.0 buffered solution to obtain a final concentration of 4.7 × 10 aM n202. Applied potential: - 0.1 V versus SCE. Immunosensor incubated for 30min in 45txl of a O.1M phosphate buffered solution at pH 7.0, containing 0.66% (v/v)

GaRIgG peroxidase conjugate and 0.1% (v/v) BSA.

sensor containing RIgG is regenerated by repeated polishing. This immunosensor has been success- ively incubated (after being polished) in a BSA solution featuring 0.63% (v/v) GaRIgG HRP conjugate as described in the Experimental section.

The relative standard deviation (RSD) in the measured current is 7% (n = 7) for a constant concentration of hydrogen peroxide of 4.7 x 10-4M. This means that regeneration by polishing of the immunocomposite is occurring. The new surface produced contains immobilized immunologic material that can interact with its antibody. Reproducibility of the response shows that the distribution of the antigen in the bulk of the composite is uniform.

Measurements in the same experimental conditions with blank sensors, based on composites but without immobilized RIgG, did not reveal peroxidase activity. Experiments demonstrated that no loss of immunological material from the immunocomposite to the solution took place.

Sandwich technique Fig. 6 shows the results using a sandwich technique for the measurement of GaRIgG (analyte). A linear relationship is noticed between log I and log CGaR~gG when the concentration of GaRIgG varied from 0 to 0.04 mg/ml (0 to 2.6 x 10 -7 M). The y-intercept was 1.88 + 0.10 and the slope is 0.43 + 0.04. The correlation is 0.990. The detection limit, defined as the concentration that produces a response three times as large as the background noise, is 9 x 10 - 12 mg/ml (5-62 × 10 17 M).

Competitive technique Fig. 7 shows the relationship between log I and log CR~gC for an immunoassay for RIgG based on the competitive technique. The linear range is between the concentrations of 0 and 0.02 mg/ml of RIgG. The y-intercept is - 0.50 + 0.25 and the slope is - 0-27 + 0.07. The correlation is 0.97. Dispersion is greater than in the sandwich technique. The assay based on the sandwich technique has a higher precision because it uses a greater relative antibody excess. Therefore, small variations of the antibody do not modify the interaction (Hoffman, 1985). This does not hap- pen in the competitive technique, where the quantity of antibody is a limiting factor which implies a lower amount of immunologic material used. The detection limit in this case is 2 × 10 -9 mg/ml (1 x 1 0 - 14M).

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Marta Santandreu et al.

1.50

1.25

1.00

0.75

0.50

0.25

0.00

-0.25

-4.5 I I I I I J

-4.0 -3.5 -3.0 -2.5 -2.0 -1.5 -1.0

log C~R~g G

Fig. 6. Calibration curve for the measurement o f GaRIgG using the sandwich technique and an RlgG- graphite-methacrylate immunosensor (for experimental details see Experimental section). Amperometric measurements of peroxidase activity were achieved by adding a standard solution of H202 to 20 ml o f 0.1 M phosphate, 1.8 x 10 - 3 M hydroquinone and 0.1 M KC1 p H 7.0 buffered solution to obtain a final concentration of 4.7 x 10 - 4 M H202. Applied potential: -0 .1 V versus SCE. Regression line: slope = 0.43 +

0.04; y-intercept = 1.88 + 0.10; r = 0.990.

M a g n e t o i m m u n o s e n s o r integrated to a f low system

The technique used here is a variation of the classic competitive technique. In the case reported, there is a preincubation between the analyte (RIgG) and an excess of the immunocon- jugate. The antigen-antibody interaction takes place in this incubation solution. This solution is injected in the flow system and transported to the magnetic solid phase containing the immobilized RIgG. The excess of labelled antibodies that had not reacted with the analyte in the first incubation interacts with the RIgG in the solid phase.

Enzymatic activity retained by the magnetic particles is measured by injecting substrate (urea) to the flow system. The generated NH3 molecules will produce a local change in pH that is detected by the ISFET.

If an NH3/NH4 + buffer is used as the carrier solution, its pH will change following the for- mula: pH = pK, + log ([NH3]/[NH4+]). If [NH4 +]

1.2

Biosensors & Bioelectronics

0.9

0.6

0 ) o

0.3

0.0

-5 -4 -3 -2 -1

log CRag G

Fig. 7. Calibration curve when measuring RIgG with a competitive technique using an RlgG-graphite-methacrylate immunosensor (for experimental details see Experimental section). Amperometrie measurements o f peroxidase activity were made by adding a standard solution of H202 to 2 0 m l of 0.1 M phosphate, 1.8 x I0 3 M hydroquinone and 0.1 M KCI pH 7.0 buffered solution to obtain a final concentration of 4.7 x ]O-aM [-I202. Applied potential: - 0.1 V versus SCE. Regression: slope = - 0.27 + 0.07; y-intercept = - 0 . 5 0

+ 0.25; r = 0.97.

is kept high and constant, pH will change linearly with respect to the logarithm of [NH3]. Therefore, pH change in the injected zone will reflect the enzyme activity. Since this is done in the context of a competitive technique, enzyme activity is inversely dependent on the concentration of the analyte in the original sample.

Reproducib i l i t y o f the response f o r di f ferent regene ra tions

The reproducibility of the immunosensor surface is particularly critical since each measurement implies the building of a new surface.

Several 200/~1 injections of fresh preincubation solutions were used to evaluate the reproducibility of the renewed surfaces. These preincubations were done by mixing 200~1 of 1.2% (v/v) GaRIgG urease conjugate and 200/~1 of 0.20 mg/ml RIgG, following the procedure described in the Experimental section. The injections were transported at a low flow rate

14


(0.02 ml/min) towards the immunosensor where the magnetic RIgG particles were immobilized. Previously, the magnetoimmunosensor had been filled by pumping 50/xl of magnetic RIgG particles suspension (1 5 mg/ml) for each assay. The urease activity retained by the magnetic RIgG particles was assessed by injecting 50/xl of 0.1 M urea. Good reproducibility was found as shown by the 2% relative', standard deviation (n = 6) (see Fig. 8).

The mechanical exchange of the immunomag- netic phase in a reproducible fashion eliminates the problems of non-stability of the immunosensing surfaces and the need for additional time, traditionally required for chemical regeneration of the used immunosurfaces, saving time and increasing sampling frequency.

Competi t ive technique Fig. 9 shows the immunosensor response to RIgG. The sensitivity obtained was - 65.96 + 6 ml/mg. The linear working range was from 0 to 0.3320 mg/ml. "['he detection limit, defined as the concentration yielding a signal three times the background noise, was 0.014 mg/ml.

105

67.5

65.0

E 62.5

60.0

57.5

55.0

70.0

i I I i i i

0 1 2 3 4 5 6

Assay number

Fig. 8. Reproducibility of the construction o f magnetic immunosensor surfaces in the flow system. The microcell was filled by pumping 50 tzl of magnetic RIgG particles (1.5 mg/ml) for each assay. Two hun- dred microlitres of equal volumes of 1.2% (v/v) GaRIgG urease conjugate and 0.20mg/ml of RIgG were injected. Preincubation time was 10 min and the incubation flow rate was 0.02 ml/min. The carrier was 0.1 M NH4CI (0.2 mb~min) and the injection volume o f

enzyme substrate (0.1 M urea) was 50 pd.

100

A

LU LU

95

90

85

80

75 I I i i i i i

0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35

RIgG (mg/ml)

Fig. 9. Calibration curve for the measurement of RIgG using a competitive technique and a flow system featuring a magnetoimmunosensor. The curve was obtained with 200 txl injections of a solution containing equal volumes of 1.2% GaRIgG urease conjugate and analyte (RIgG) solution of different concentrations. Preincu- bation time was lOmin and the incubation flow rate was 0.02 ml/min. The carrier solution was NH4C1 0.1 M (0.2 ml/min), the enzyme substrate was 0.1 M urea and injection volume 50 txl. The immunosensor was built by injecting 50 txl of magnetic RIgG particles (1.5 mg/ml). Regression: slope = - 66 + 6; y-intercept = I00 + 1;

r = 0.998.

CONCLUSIONS

The electrochemical immunosensor systems developed allow the renewal of the immobilized immunologic material on the sensitive surface of the transducer. These permit repeated use of the reported immunosensors since the renewed surface and the resulting electrochemical signals are reproducible.

Immunocomposites are ideal for manual assays. Furthermore, these materials are technologically attractive since they can be built easily and are mouldable before curing, which enables the pro- duction of sensors of different shapes and sizes. After curing, they are rigid, so their surfaces can be altered mechanically. The contents of the components are controlled easily and are compat- ible with the blended biomaterial. The distribution and the characteristics of the conducting particles produce an enhanced electrochemical response (microelectrode array behaviour).

15


In the case of the magnetoimmunosensors, the automation of a conventional immunoassay technique has been possible, since it is based on a compact immunosensor with renewable surface. The use of magnetic immunoparticles obviates the need for the chemical regeneration of the immunosensing surface. This produces a better reproducibility and reduces the analytical cycle. Several key elements of the assay are integrated in a single area of the flow system, namely the flow cell. In this microcell there is a confluence of the antigen-antibody reaction, the detection of the signal and the application of the magnetic field. This approach results in higher sensitivity and a shorter response time, owing to the proximity between the immunoparticles and the sensing surface.

The magnetoimmunosensor system reported here is more compact and robust than other flow systems featuring immunoreagents immobilized on magnetic particles but based on magnetoim- munoreactors not integrated to the sensor.

ACKNOWLEDGEMENTS

M. S. acknowledges the support received from the Comissionat per a Universitats i Recerca of the Generalitat of Catalonia (FI/95-2117). This work has been funded by the European Com- mission, Directorate General for Science, Research and Development (XII-D-1), Environ- mental Technologies (EV5V-CT94-0407), the Comisi6n Interministerial de Ciencia y Tecnol- ogfa, Spain (BIO95-1196-CE and BIO96-0740) and the Grupo de Ayuda de la Microelectr6nica Espafiola (GAME). Ilchmann et al., 1990 are also acknowledged.

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Development of electrochemical immunosensing systems with renewable surfaces

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