Development of piezoelectric crystal based microgravimetric immunoassay for determination of insulin concentration

journal of

Motcchnology ELSEVIER

Journal of Biotechnology 39 (1995) 27-34

Development of piezoelectric crystal based microgravimetric immunoassay for determination of insulin concentration C. Raman

Suri a, P.K. Jain b, G.C. Mishra a, *

a Institute of Microbial Technology, Sector 39-A, Chandigarh-160 014, India b Central Scientific Instruments Organisation, Sector 30, Chandigarh-I60 020, India Received 29 September 1994; accepted 26 October 1994

Abstract A microgravimetric, piezoelectric crystal based immunoassay for the quantification of insulin concentration is described. The method utilizes a modified piezoelectric crystal device having an antibody specific to insulin bound to its surface. The antibody to insulin was immobilized on the surface of crystal electrode by using either 3-aminopropyltriethoxy silane (3-APTES), polyethyleneimine (PEI) or covalently coupled protein A-gold immobilization method. Coating an electrode with a cross linked protein A-antibody complex gave better results in terms of sensitivity and stability. Using the system described, the insulin concentration up to 1 ng ml-’ could be detected. The stability and reusability of the system was further improved by using a mild eluting reagent which successfully removed the bound insulin molecules from the antibody-coated crystal without affecting the immobilized insulin antibody. Scanning tunneling microscopic (STM) study was also done to confirm the surface coverage and orientation of insulin and antibody molecules on the modified piezoelectric crystal electrode surface. A comparison between the present study and the well-established radioimmunoassay technique (RIA) revealed that the described microgravimetric im-

munoassay technique (MIA) could successfully be developed as an alternative of RIA. Keywords:

Microgravimetric

immunoassay;

Piezoelectric crystal device; Radioimmunoassay;

1. Introduction

Estimation of insulin concentration in serum is becoming increasingly important for the differentiation and diagnosis of various types of diabetes (Pierluissi and Campbell, 19801, and its determination is recommended in the evaluation of patients with chronic pancreatitis. The well-estab-

* Corresponding

author.

Elsevier Science B.V. SSDI 0168-1656(94)00136-7

Protein A; Insulin

lished radioimmunoassay (RIA) technique has been widely applied for the measurement of insulin level in serum (Wilson and Miles, 1977; Yalow, 1980; Dron et al., 1980). However, this technique suffers from a series of drawbacks such as, complexity in setting up the experiment, usage of hazardous radioactive materials and time-consuming work. Efforts have also been made to develop an enzyme based biosensor using glucose oxidase as catalyst for monitoring blood glucose level (Turner and Pickup, 1985; Shaw et al., 1991) correlating the concentration of insulin in blood.

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of Biotechnology

It is found that insulin secretion by p-cells is extremely high in response to glucose present in circulation (Reach and Wilson, 1992). Because of this rapid response, this method of estimation of insulin concentration is not yet standardized and needs to be improved. In addition, this method is still not very sensitive and stable. Hence, there is a strong need for development of an alternative, sensitive, recyclable technique which can reliably measure insulin concentration. A microgravimetric immunoassay technique using piezoelectric crystal device as a sensing element has earlier been employed for the estimation of different biomolecules (Shons et al., 1972; Roederer and Bastiaans, 1983; Ngeh-

39 (1995) 27-34

Ngwainbi et al., 1990). This technique is relatively simple, reliable and does not require the usage of hazardous radioactive materials. The immunoassay technique is based on the measurement of small changes of mass, resulting from formation of antibody-antigen complex. The surface mass change (6m) and resonant frequency shift (6F) of a piezoelectric crystal vibrating at fundamental frequency (F) was described by Eq. 1 (Sauerbrey, 1959) 6F=

-kFcf6m

= -2.26

XA-’

X 10-6F~6m

XA-’

(1)

where SF is the change in frequency due to the coating (Hz), k is proportional constant depend-

-

[ -

b

(b)

Cl f

Vcc

c2

V cc 1

ECL *“TP”T

t

V cc

-OUTPUT

PZ - Crystal

Fig. 1. (a) Block diagram of piezoelectric crystal immunobiosensor system (a) air temperature bath, (b) piezoelectric crystals (sample and reference) in detector cell designed for its optimum temperature, humidity and air flow rate conditions, Cc) oscillator circuit Cd) S-digit frequency counter and (e) a mixer, to give difference output of two frequencies (sample and reference). (b) Piezoelectric crystal oscillator, incorporating (a) voltage regulator amplifier. (h) automatic gain control, (c) emitter coupled logic output and Cd) transistor-transistor logic output.

C.R. Suri et al. /Journal

of Biotechnology 39 (1995) 27-34

ing upon density and shear modulus of quartz crystal (for AT-cut quartz, the density is 2.648 g cme3 and shear modulus is 2.947 X lOi’ dynes per cm2), F, is the fundamental frequency of the quartz plate (MHz), 6m is mass of deposited coating (g) and A is coated area (cm2>. The use of piezoelectric crystal based immunobiosensor to demonstrate the antigen-antibody complex on its modified surface is now been attempted for the estimation of biomolecules. An improved piezoelectric crystal based immunoassay method for the determination of antigens was described by Oliveira and Silver (1980). An ATcut crystal modified with anti-human IgG, which showed a change in resonance frequency on binding to human IgG was demonstrated by Thompson et al. (1986). Muramatsu et al. (1987) also demonstrated that a layer of protein A immobilized on the surface of piezoelectric crystal could be successfully utilized to determine the concentration of immunoglobulin (IgG) and its subclasses in solution. In this communication, we report the design and development of a specific and stable piezo immunobiosensor for detection of insulin concentration in solution. Different immobilization methods (Guilbault et al., 1992) were used to study the sensitivity and stability of the developed detector system.

2. Materials and methods Piezoelectric crystals (AT-cut) with a resonant frequency of 10 MHz were obtained from Universal Sensors Inc., USA. Anti-human insulin antibody was a kind gift from AIIMS Delhi. Insulin (porcine, specific activity 26 U mg- ‘I and protein A were purchased from Sigma Co. USA. Bovine serum albumin (BSA), 3-aminopropyltriethoxysilane (3-APTES), glutaraldehyde, polyethyleneimine and dimethyl pimelimidate (DMP) were obtained from Aldrich Chemical USA. All buffers and solutions were made up of particle-free distilled Milli-Q water. The composition of the buffers are as follows: buffer A phosphate buffer (50 mM, pH 7.2); buffer B phosphate buffer saline (20 mM, 150 mM NaCl, pH 7.0); buffer C - phosphate buffer saline (20

29

mM, 150 mM NaCl, pH 7.4); buffer D - glycineHCl (100 mM, pH 2.8). Gentle antibody-antigen eluting buffer (pH 6.9) was obtained from Pierce Co., USA. All other reagents and solvents used were of analytical grade commercially available. 2.1. Immunobiosensor kit A highly stable and compact piezoelectric crystal based immunobiosensor system (Suri et al., 1994) was used for the estimation of insulin concentration in this study (Fig. la). The system consisted of a stable oscillator circuite (Fig. lb), modified piezoelectric crystal device, frequency monitor and a differentiating circuit. The crystals used in this study were stable AT-cut quartz crystals (AT refers to the angle of cut of quartz plate which is 35” 15’ for AT quartz crystal). The whole oscillator circuit was kept inside a shielded box to reduce the stray parallel capacitance and unwanted interferences from external sources. The frequency stability of the developed immunobiosensor was 1 Hz during 6 h observation time. 2.2. Immobilization procedure Prior to biomolecules immobilization on the piezo crystal, the surface was thoroughly cleaned (degreased) in a sonicator bath for 2 min and further treated with a 10% hydrofluoric acid to increase the surface area (Roederer and Bastiaans, 1983). The crystals were then chemically modified for ligand binding by following immobilization methods mentioned below. Method 1 (3-aminopropyltriethoxysilane method)

The crystals were first treated with 3-APTES solution (5% in dry acetone) for 2 h at room temperature and then washed with the same solvent. The modified crystals were further dried in a vacuum oven for 90 min at 110°C to enhance the binding of 3-APTES molecules on the surface. The silanized crystals were again dipped in a 2.5% glutaraldehyde solution in buffer A for 2 h and then washed several times with same buffer to remove the excess glutaraldehyde. Subsequently, 20 ~1 of 0.5 mg ml- ’ antibody solution

Xl

C.R. Suri et (11./Journal

ofBiotechnology 39 (1995) 27-34

was placed on the crystal electrodes and left overnight at 4°C. After incubation, the remaining unreacted aldehyde was blocked with 0.1 M glycine solution in buffer B, followed by rinsing with distilled water. The steady resonant frequency was measured after drying the crystals. Method 2 (polyethyleneimine method) The crystals were immersed in a polyethyleneimine solution (2% in methanol) for 30 s. After air drying, the crystals were washed several times with same solvent to remove excess unbound material. This was followed by immersing the crystals in a 2.5% glutaraldehyde solution in buffer A for 2 h at room temperature and then washed with distilled water. Subsequently, 20 ~1 of insulin antibody solution was placed on both sides of crystal electrodes and kept for 1 h at room temperature. Unreacted aldehyde groups were blocked by immersing the crystals in a solution of 0.1 M glycine in buffer B. The crystals were washed again with distilled water, dried and steady resonant frequency was measured. Method 3 (protein A-gold immobilization method) The crystals were dipped in 1.2 N NaOH for 30 min and washed with distilled water. The crystals were further soaked in 1.2 N HCl for 5 min and thereafter 50 ~1 of 12 N HCl was placed on both sides of the electrodes for 2 min. The crystals were thoroughly washed with distilled water followed by ethanol and dried at 70°C for 30 min. They were then dipped in protein A solution (1 mg ml-’ in buffer A) and incubated for 2 h at room temperature. After washing with same buffer, the coated crystals were immersed in BSA solution (1 mg ml ~ ’ in buffer A) to block all nonspecific protein binding sites, then were further washed with same buffer and, subsequently, 20 ~1 of anti-insulin antibody was added to the crystal electrodes and left overnight at 4°C. Anti-insulin antibody was then covalently coupled to the protein A-coated crystals by dipping the crystals in DMP solution (5 mg ml-’ in 0.2 M triethanolamine, pH 8.2) for 1 h at room temperature with gentle shaking. They were then washed with distilled water and dipped in ethanolamine solution (0.1 M, pH 8.2) to block any remaining

active sites of the cross linker. Crystals again washed with distilled water, dipped M NaCl and then in 0.1 M glycine-HCl (buffer D) to remove noncovalently bound body from the protein A-coated surface. washing with distilled water, the crystals dried in clean air and the steady resonant quency was measured.

were in 0.5 buffer antiAfter were fre-

2.3. Scanning tunneling microscopic (STM) imaging The STM imaging of insulin and antibody to it was carried out by using Nanoscope 11 (Digital Instruments Inc., USA). STM in air with platinum-iridium tip. Anti-insulin antibody was immobilized on gold electrode of piezo crystals as described in our earlier study (Suri et al., 1994) using protein A-gold immobilization method. Insulin molecules were bound on antibody-coated surface by antibody-antigen reaction. 2.4. Estimation of insulin by microgravimetric immunoassay The antibody-coated crystal (sample) was dipped into a known concentration of several different insulin solutions in buffer A so as to enable the formation of antibody-insulin complex on the crystal electrode surface. Crystal was then rinsed in 0.5 M NaCl followed by distilled water to remove any nonspecific binding. After washing, the coated crystal was air dried and steady resonant frequency was measured. The frequency differences between antibody-coated crystal and insulin bound to antibody-coated crystal was calculated and correlated with the concentration of insulin. For successive measurement of insulin concentrations, insulin bound to antibody-coated crystal was removed by using a mild eluting reagent from Pierce, Co., USA. 2.5. Estimation of insulin by radioimmunoassay Insulin concentration was also measured by RIA (Yalow, 1980) method, using a standard RIA kit (RIAK-1) supplied by BARC, India.

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of Biotechnology 39 (1995) 27-34

3. Results and discussion In the present study, we have coated insulin antibody on gold electrodes of the piezoelectric crystals using three different immobilization methods. The main concern with conventional methods of antibody immobilization in this type of study is (a) the stability of the antibody immobilized on matrix and (b) availability of free antigen binding sites. Protein A immobilized on gold surface has been successfully used to orient the antibody with its antigen binding sites sterically free for antigen binding. Upon cross linking the protein A-antibody complex with DMP creates a highly stable support with excellent retention of antigen binding activity (Schneider et al., 1982). We have found that the best results in terms of long-term sensitivity and stability were obtained when the antibody was covalently coupled to the surface via protein A-gold immobilization method. Glutaraldehyde used in 3-APTES and PEI immobilization methods, adversely affected the gold electrode, because of its high reactivity with the surface leading to an overall reduction in the stability of the system (Guilbault and Luong, 1989). The commercial preparation of glutaraldehyde also quickly deteriorated to unsuitable polymeric products (Barnes et al., 1991). On the other hand, protein A-gold complexes are very stable and have an association constant of lo8 M-’ (Roth, 1982). The deposition of protein A on 10 MHz AT-cut piezoelectric crystal resulted in approx. 200 Hz decrease in resonant frequency of the crystal, which is in agreement with the work of Davis and Leary (1989). Piezoelectric crystal coated with protein A binds to the Fc region of the insulin antibody which further resulted in decrease of resonant frequency. The saturation of the crystal frequency occurred between 0.5 mg ml- ’ and 1.0 mg ml-’ antibody concentration. The frequency shift did not increase significantly with further increase in antibody concentration (Fig. 2). The anti-insulin antibody-coated crystal was then used for the determination of insulin concentration. In our study, it was found that the antibody-insulin complex formation on piezoelectric crystal surface became stable after 60 min incubation time

3 E

250

,

225

-

31

x

150 0.0

I

I

I

0.5

1.0

1.5

Antibody

Concentration

(me

2.0

ml-‘)

Fig. 2. Response of different concentration of insulin antibody to the protein A modified crystal surface. Each point represents the average of three experiments.

at room temperature (Fig. 3). Increasing the equilibration time, however, did not change the resonant frequency to any significant value. In order to determine the sensitivity and accuracy of the present immunoassay, piezoelectric crystals coated with insulin antibody were dipped in different concentrations of insulin solution in buffer C. A linear correlation was observed between 1 ng ml-’ to approx. 0.1 mg ml-’ of insulin concentration as a function of resonant 150

125

-

100

-

75

-

3 " 2 8 6 9 $ t:

50 ' 0

I

I

I

30

60

90

I

J

120

150

Time (min)

Fig. 3. Time resonance curve of the insulin antibody reaction with insulin antigen. The reaction completed (saturated) after about 60 min at room temperature. Additional time did not give any significant change in the frequency response.

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C.R. Suri et ul. /Jourmd

of Biotechnology 39 (1995) 27-34

150 r

T

125

i’

,/!

,’

+

1 0

?

4

Number

Fig. 4. The dependence of insulin antibody immobilized immunobiosensor system on the concentration of insulin antigen. The antibody concentration was 0.5 mg ml- ‘. Each point represents the average of three experiments.

frequency shift (Fig. 4). Above 0.1 mg ml ~’ of insulin concentration, the resonant frequency shift saturated gradually, probably due to the binding limit of the antibody-coated surface. The log [concentration of insulin] vs. mean frequency shift was fitted using linear regression method. Not much variation was observed between the percent variation of actual and computed frequencies which confirms the sensitivity of the presented assay about 1 ng ml ’ of insulin concentration. The reusability and long-term stability of the insulin antibody-coated crystal immobilized via protein A-gold method is depicted in Fig. 5. Bound insulin was removed from the antibodycoated crystal by using dissociating agents such as 0.1 M glycine-HCl (buffer D>, and immunopure gentle antibody-antigen (Ab/Ag) eluting buffer (pH 6.9) from Pierce Co., USA. In this study, the reagent obtained from Pierce worked satisfactorily. Removing the bound insulin from the complex using buffer D affected the antibody bound on the protein A-coated crystal. In contrast, Pierce immunopure gentle Ab/Ag eluting buffer successfully eluted the bound insulin from the antibody coupled matrix without affecting antibody (data not presented). By using this eluting reagent we have successfully demonstrated the

6

8

10

12

of Assays

Fig. 5. Correlation between the regeneration cycle (number of assay) of insulin antibody-coated crystal with different eluting buffers (a) 0.2 M glycine HCI (0) and (b) Pierce immunopure gentle antibody-antigen eluting buffer (01. The graph shows that the insulin antigen eluted with Pierce eluting buffer gave maximum number of assays.

regeneration of the coated crystal for at least nine to ten assays. The STM image of insulin antibody immobilized on crystal electrode surface using protein A-gold immobilization method is shown in Fig. 6. From this figure it can be seen that the antibody (immobilized at 0.5 mg ml-’ concentration) uniformly covered the crystal surface without aggre-

Fig. 6. STM image of insulin antibody immobilized on crystal electrode surface using protein A-gold immobilization method. Antibody concentration is 0.5 mg ml ‘.

C.R. Sun’ et al. /Journal

of Biotechnology 39 (1995) 27-34

33

temperature, humidity, drying of crystal, etc., in MIA. However, there is considerable scope for improving the experimental design to enhance the performance of the assay, which is underway in our laboratory.

4. Conclusions

Fig. 7. STM image of insulin molecules bound to insulin antibody coated surface (STM bias voltage -969.5 mV, tunneling current 0.25 nA and scan rate 19.69 Hz.). Insulin concentration is 0.1 mg ml-‘. Insulin molecules are seen as cylindrically shaped with apparent dimensions of about 18.37 X71.91 nm.

gates formation. Hence, the concentration of insulin antibody was therefore fixed at 0.5 mg ml-’ for all experiments. The STM images of insulin molecules bound to antibody-coated surface have a closely packed and uniformly oriented insulin molecules on the crystal surface (Fig. 7). The insulin molecules were found to be cylindrical in shape with apparent dimensions of individual molecules of about 18.37 x 70.91 nm. The increase in size of insulin (immobilized) molecules as compared with X-ray crystal structure data (50 x 35 A) observed by Arnebrant and Nylander (1988) is due to the presence of remaining solvent around the insulin molecules after immobilization. The results obtained for the measurement of insulin concentration by employing the above-described microgravimetric immunoassay (MIA) are comparable to those obtained by RIA. As a check, a standard insulin solution (52 pIJ per mg> was measured by MIA as well as RIA methods. The results obtained in the present study were in reasonable agreement with the RIA method. Minor differences observed between the two methods can arise from half-life of radioactive materials used in RIA, and environmental factors like

We have developed a microgravimetric immunoassay kit using piezoelectric crystal as detector system for estimation of insulin concentration. The detector system has been extensively tested for its long-term stability and sensitivity. The results obtained from three immobilization methods demonstrated that the covalently coupled antibody on protein A-gold surface can be successfully employed for microgravimetric immunoassay. Despite the very promizing results in the present study, a number of unknown parameters, such as antibody-antigen binding affinities, acoustic properties of adsorbents and viscoelastic responses are to be understood and implemented before such assay is used as routine immunoassay for insulin estimation. The future use of this technique described in this report is thus very promizing.

Acknowledgements

Authors are also grateful to Dr. R.P. Gupta (CEERI, Pilani) for providing STM facility and Dr. Maria from Pierce Co., USA for suggesting the use of DMP in this experiment. We are equally thankful to Dr. Krishna Sastry for critical reviewing the manuscript.

References Arnebrant, T. and Nylander, T. (1988) Adsorption of insulin on metal surfaces in relation to association behavior. J. Colloid Interface Sci. 122, X7-565. Barnes, C., D’Silva, D., Jones, J.P. and Lewis, T.J. (1991) A concanavalin-A coated piezoelectric crystal biosensor. Sensors and Actuators B 3, 295-304. Davis, K.A. and Leary, T.R. (1989) Continuous liquid phase

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piezoelectric biosensor for kinetic immunoassay. Anal. Chem. 61. 1227-1230. Dron, D.I., Jones, R.H., Sonksen, P.H., Thomas, J.H. and Brandenburg, D. (1980) Radioimmunoassay of chemically modified insulin. Diabetologia 18, 59-63. Guilbault. G.G. and Luong, J.H.T. (1989) Immobilization methods for piezoelectric biosensor. Biotechnology 7. 349-35 1, Guilbault, G.G.. Schmid, R. and Hock, B. (1992) Piezoelectric immunobiosensor for atrazine in drinking water. Biosensor and Bioelectronics 7. 41 l-419. Muramatsu, H., Dicks, J.M., Tomia, E. and Karube, I. (1987) Piezoelectric crystal biosensor modified with protein A for determination of immunoglobulins. Anal. Chem. 59, 27602763. Ngeh-Ngwainbi, J.. Suleiman, A.A. and Guilbault, G.G. (1990) Piezoelectric ctystal biosensors. Biosensor and Bioelectronics 5, 13-26. Oliveira, J. and Silver, S.F. (1980) US Patent 424096. Pierluissi, J. and Campbell, J. (1980) Metasomatotrophic diabetes and its induction. Diabetologia 18, 223-228. Reach, G. and Wilson, G.S. (1992) Can continuous glucose monitoring be used for the treatement of “Diabetes”? Anal. Chem. 64, 381A-386A. Roederer, E. and Bastiaans, G. (1983) Microgravimetric immunoassay with piezoelectric crystals. Anal. Chem. 55, 2333-2336. Roth, J. (1982) Evalution of the protein A-gold (pAG) technique for labelling of multiple antigens : pAG complexes

with 3 nm and 15 nm gold particles. Histochem. J. 14, 791-801. Sauerbrey, G. (1959) The use of oscillators for weighting thin layers and for microweighing. Z. Phys. 155, 226. Schneider, C., Newman, R.A., Sutherland, D.R., Asser, U. and Greaves, M.F. (1982) A one step purification of membrane proteins using a high efficiency immunomatrix. J. Biol. Chem. 257, 10766-10769. Shaw, G.W., Clavemont, D.J. and Pickup, J.C. (1991) In vitro testing of a simply constructed, highly stable glucose sensor suitable for implantation for diabetes patients. Biosensors and Bioelectronics 6, 401-406. Shons, A., Dorrman, F. and Najarian, J. (1972) An immunospecific microbalance. J. Biomed. Mater. 6, 565-570. Suri, C.R., Raje, M. and Mishra, G.C. (1994) Determination of immunoglobulin M concentration by piezoelectric crystal immunobiosensor coated with protamine. Biosensor and Bioelectronics 9, 325-332. Thompson, M., Arthur, C.L. and Dhaliwal, G.K. (1986) Liquid phase piezoelectric and acoustic trans studies of interfacial immunochemistry. Anal. Chem. 58, 1206-1209. Turner, A.P.F. and Pickup, J.C. (1985) Diabetes Mellitus: Biosensor for research and management. Biosensors 1, 85-l 1s. Wilson, M.A. and Miles, L.E.M. (1977) Radioimmunoassay of insulin. In: Abraham, G.E. (Ed.), Handbook of Radioimmunoassay, Marcel Dekker, Inc., New York, pp. 275-279. Yalow, R.S. (1980) Radioimmunoassay. Annu. Rev. Biophys. Bioeng. 9, 327-345.