Label-free Detection of p53 Antibody Using A Microcantilever Biosensor With Piezoresistive Readout

Youzheng Zhou1, Zheyao Wang1, Wentao Yue2, Kai Tang2, Wenzhou Ruan1, Qi Zhang1, and Litian Liu1
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Institute of Microelectronics, Tsinghua University, Beijing 100084, CHINA 2 Beijing Chest Hospital, Beijing 101149, CHINA Email: z.wang@tsinghua.edu.cn by detecting DNA mismatch or protein biomarkers [3]-[5]. The detection is normally achieved by modifying the surface of the microcantilever with recognition molecules. The specific interaction between the recognition molecules and the target biomarkers generates surface stress in microcantilever and in turn the deflection of the microcantilever. The deflection, which is normally several nanometers, can be measured using either optical or piezoresistive readout. Due to the importance of p53 antibody in cancer diagnosis, we take the advantage of microcantilever biosensors to specifically translate the molecular recognition into nanomechanical response. It is achieved by immobilizing p53 antigen on the surface of the microcantilever as recognition probe to detect p53 antibody, the target biomarker, by measuring the deflection of the microcantilever, which is caused by the changes of the surface stress as a result of the specific bioaffinity between the antigen and the antibody. The deflection is measured using integrated piezoresistors, which eliminates the bulky and expensive apparatus in common used optical readout. Quantitative detection of p53 antibody ranging from 20ng/ml to 20g/ml has been achieved. Compared with conventional ELISA or immunofluorescene assay, the present method has the advantages of label-free operation, fast detection, and low cost. II. EXPERIMENTAL

AbstractWe report the detection of p53 antibody as a biomarker for early-stage cancer diagnosis using a microcantilever biosensor with piezoresistive readout. The accumulation of p53 antibody in human sera is found strongly involved in a variety of human cancers, thus simple and fast detection of the level of p53 antibody is of importance in clinical application. In this work, p53 antigen is immobilized on the surface of the microcantilever as a recognition probe to detect p53 antibody by measuring the deflection of the microcantilever using integrated piezoresistors, which is caused by the changes of the surface stress as a result of the specific bioaffinity between the antigen and the antibody. Quantitative detection of p53 antibody ranging from 20ng/ml to 20g/ml has been achieved. Compared with conventional ELISA or immunofluorescene assay, the microcantilever sensor has the advantages of labelfree operation, fast detection, and low cost.

I.

INTRODUCTION

Diagnosis and prognosis of cancer patients with earlystage detection of convincing biomarkers is crucial for better treatment and higher survival rate of the patients. During the past decades, p53 gene, which is involved in regulating cell growth, replication and apoptosis, has been proved as one of the most important tumor suppressor by sensing DNA damage and facilitates repair. Extensive studies have shown that the mutation of p53 gene is found to associate with a wide variety of malignant tumors including breast, lung, prostate, ovarian and melanoma [1][2]. The mutation results in the function alteration of p53 protein from growth regulation to rapid proliferation of cells and accumulation of p53 protein in nuclei of tumor cells. The accumulation of p53 protein further induces a higher level of p53 antibody in the serum of cancer patients through immune response, which has been found to be positively correlated with poor survival rate of the patients. Thus p53 antibody serves as a highly specific and effective clinical biomarker for early-stage cancer diagnosis and prognosis. Several conventional biochemical analytical methods such as enzyme-linked immunosorbent assay (ELISA) or immunofluorescene assay have been applied to detect p53 antibody. Each method has its inherent advantages and disadvantages. Microcantilevers have been emerging as sensitive and label-free diagnostic platform for malignant disease diagnosis
This research is supported in part by the NFSC under Grant 6040009 and 863 Program under Grant 2007AA03Z04.

A. Microcantilevers The microcantilever sensor presented here uses the integrated piezoresistors fabricated in the single crystalline device layer of silicon-on-insulator (SOI) wafer, which can achieve larger piezoresistive coefficients and higher sensitivity than polycrystalline silicon piezoresistors. Fig.1 shows the schematic illustration of the cross-section of the microcantilever. The geometrical and fabrication parameters are optimized to increase the sensitivity of surface stress change in protein detection [6]. The fabrication of microcantilever starts with a SOI wafer with 100nm-thick ptype (100) top device layer and 400nm-thick buried oxide layer, and the detailed process is published elsewhere [7]. The piezoresistors are formed by completely removing top device layer at other regions, and are fully encapsulated in silicon dioxide dielectric layer and buried oxide layer to avoid current

978-1-4244-5335-1/09/$26.00 2009 IEEE

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leakage and contamination during detection. Thin and uniform microcantilevers are suspended using a two-step front-side releasing method to achieve high efficiency and high yield [8]. Owing to the total microcantilever thickness of 650nm and the lower Youngs modulus of silicon dioxide, the sensitivity of the present microcantilever is much larger than the former design [8]. Fig.2(a) shows the SEM image of a fabricated piezoresistive microcantilever, and Fig.2(b) shows optical photo of the piezoresistor on the microcantilever.

cantilever and the other as a reference cantilever. p53 antigen is only immobilized on the sensing cantilever, but not on the reference cantilever, thus the specific interaction is localized. The piezoresistor changes caused by the deflection are readout using an on-chip integrated Wheatstone bridge configuration, which consists of four piezoresistors with nominal identical resistance value, i.e. two piezoresistors on the differential cantilevers and two piezoresistors on substrate, as shown in Fig.2(b). During the detection, the output signal of the Wheatstone bridge is amplified and filtered using an off-chip instrumental amplifier. B. Functionalization The microcantilevers are first modified using the following procedures to immobilize p53 antigen before detection. Covalent binding of recognition molecules onto solid surface by self-assembly monolayer (SAM) is usually used as an effective and reliable immobilization method. 5nm chromium and 30nm gold are successively deposited on the upper surface of the sensing cantilever, but not on the reference cantilever. After cleaned in acetone, ethanol and deionized water for 10min, the microcantilevers are thoroughly rinsed in an ethanol solution of 1mM 11-mercaptoundecanoic acid (11MUA) for 12h. The mercapto-group (-SH) of 11-MUA SAM can covalently bind on the gold surface compactly and spontaneously, and the terminal carboxy-group (-COOH) can covalently bind with the residual amino-group (-NH3) of p53 antigen by condensation reaction, thus it acts as a bridge to immobilize the p53 antigen onto microcantilevers.

Fig.1. Schematic illustration of the microcantilever with piezoresistive readout.

(a)

Microcantilever Lateral etching Piezoresistor

Fig. 3. Schematic view of the sensing microcantilever after functionalisation.

Aluminum Contact hole

(b) Fig.2. SEM image of: (a) a fabricated piezoresistive microcantilever and, (b) an on-chip piezoresistor and Wheatstone bridge configuration.

To eliminate the common mode errors such as self-heating and vibration, two adjacent microcantilevers are operated in differential mode during detection, i.e., one as a sensing

The terminal carboxy-group of 11-MUA SAM is activated by rinsing in the coupling reagents of 0.4M 1-ethyl-(3dimethylaminopropyl) carbodiimide hydrochloride (EDC.HCl) and 0.1M N-hydroxysulfo-succinimide (NHS) for 30min to form NHS-ester [9]. Then the microcantilevers are cleaned in deionized water thoroughly to remove undesired chemicals, and the p53 antigen is then covalently immobilized by incubating in a solution of 100g/ml p53 antigen in sodium phosphate buffer (PBS, 0.01M, pH=7.4) for 3h. The NHSester is replaced by the residual amino-group of p53 antigen to complete the condensation reaction. To prevent unspecific binding of biomarkers on the surface of the reference cantilever during detection, the microcantilevers are rinsed in 15mg/ml bovine serum albumin (BSA) in PBS for 1h. After

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that, the microcantilevers are rinsed exhaustively in PBS to remove redundant protein molecules before detection. The functionalized sensing microcantilever is schematically illustrated in Fig.3. The results of the SAM and the immobilization of the recognition proteins are validated using fluorescence imaging. Before p53 detection, the microcantilever sensor is first characterized using the bioaffinity of IgG antigen and antibody, as IgG protein pairs have been already known to generate surface stress changes. The functionalization procedure follows the method aforementioned, and the IgG antibody is immobilized on the surface of the microcantilever to detect IgG antigen. After functionalization, the p53 antibody detection experiment is performed in a liquid chamber. The microcantilever surface is modified using the aforementioned method, and before use the microcantilevers are stabilized in PBS until the output voltage of the Wheatstone bridge achieves equilibrium state. Then different p53 sample reagents with known concentration of p53 antibodyare injected into the chamber with a flow rate of 100l/min, and the real-time voltage changes are recorded and are translated into surface stress changes of the microcantilevers. III. RESULTS AND DISCUSSION

linear. This verifies the function of bioaffinity detection of the microcantilevers, and then they are used to detect p53 antibody. Before p53 detection, BSA with concentration of 15mg/ml is first injected as a control experiment to verify the specificity of the microcantilever. It can be seen that such a high concentration BSA produces a small surface stress change of 2mN/m, which is negligible when compared with other specific bioreactions. This indicates that no specific binding occurs between p53 antigen and BSA.

Concentration of anti-rabbit IgG(ng/ml)

Fig.5 The relative photoresistor changes versus the antibody concentration.

Fig.4 shows a fluorescent photo of a glass carrier to verify the SAM and the immobilization of antigen. The top left corner is the photo activated using 492nm laser, the top right corner is the photo obtained under natural light, and the bottom photo is the combination of the two above. It can be seen that the surface treated using EDC.HCl and NHS emits green fluorescent light, whereas there is no fluorescent light for the area without gold. This proves that the antigen is immobilized on the gold surface.

Then four functionalized microcantilever sensors are used to detect p53 antibody samples with different concentrations, namely 20ng/ml, 200ng/ml, 2g/ml, and 20g/ml. The measurement results are shown in Fig.6. It can be seen that the injection of p53 samples impacts the microcantilever, and a pulse voltage occurs for a very short period (about 10s) before the voltage restores. For each concentration, the surface stress increases with time and reaches a saturation stage in several thousand seconds. The time to saturation is concentration dependent, i.e., high concentration takes more time than lower ones. Compared with the BSA control experiment, it can be concluded that the changes in the surface stress of the microcantilevers are caused by the specific binding between p53 antigen and antibody, and the microcantilevers bend downwards. The results show that the surface stress changes increase with the increase of p53 antibody concentration, which means that quantitative detection of p53 antibody can be accomplished in less than 20 minutes. The experiment also gives that the limit of the detection is about 20ng/ml, which is determined by the fluctuation of the output signal. When p53 antibody with concentration of 2ng/ml is introduced, the output voltage can be distinguished from that induced by unspecific BSA. The fluctuation and the deviation of the results mainly originate from the environmental noise and variance of the process parameters among the microcantilevers. Therefore, in order to improve the limit of detection, further adjustment of fabrication parameters and depression of environmental noise have to be done.

Fig.4 Fluorescent optical photo of the modified surface of the microcantilever

Fig.5 shows the relative changes in the resistance versus the concentration of IgG antigen induced by and binding between IgG antigen and antibody. It can be seen that the specific bioaffinity of IgG antibody and antigen bends the microcantilever, and the resistance changes increase with the concentration of IgG antigen, although the relationship is not

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in the applications of rapid and simple diagnosis of early-stage cancers. REFERENCES

[1] M. Hollstein, D. Sidransky, B. Vogelstein, and C.C. Harris, p53 mutations in human cancers, Science, vol. 253, pp. 4953, 1991. [2] O. J. Finn, Immune response as a biomarker for cancer detection and a lot more, New Engl. J. Med., vol. 353, pp. 128890, 2005. [3] J. Fritz, M.K. Baller, H.P. Lang, H. Rothuizen, P. Vettiger, E. Meyer, et al., Translating biomolecular recognition into nanomechanics, Science, vol. 288, pp. 316318, 2000. [4] G. Wu, R.H. Datar, K.M. Hansen, T. Thundat, R.J. Cote and A. Majumdar, Bioassay of prostate-specific antigen (PSA) using microcantilevers, Nat. Biotechnol., vol. 19, pp. 856860, 2001. [5] K.W. Wee, G.Y. Kang, J. Park, J.Y. Kang, D.S. Yoon, J.H. Park and T.S. Kim, Novel electrical detection of label-free disease marker proteins using piezoresistive self-sensing micro-cantilevers, Biosens. Bioelect., vol. 20, pp.193238, 2005. [6] Z. Wang, Y. Zhou, C. Wang, Q. Zhang, W. Ruan and L. Liu, A theoretical model for surface-stress piezoresistive microcantilever biosensors with discontinuous layers, Sens. Actuat., B: Chemical, vol. 138, pp. 598606, 2009. [7] Y. Zhou, Z. Wang, C. Wang, W. Ruan and L. Liu, Design, fabrication, and characterization of a two-step released silicon dioxide piezoresistive microcantilever immunosensor, J. Micromech. Microeng., vol. 19, 065026, 2009. [8] Y. Zhou, Q. Zhang, Z. Wang and L. Liu, A silicon microcantilever biosensor fabricated on SOI wafers using a two-step releasing approach, IEEE Sensors 2008, pp. 227230. [9] N. Patel, M.C. Davies, M. Hartshorne, R.J. Heaton, C.J. Roberts, et al., Immobilization of protein molecules onto homogeneous and mixed carboxylate-terminated self-assembled monolayers, Langmuir, vol. 13, pp. 64856490, 1997. [10] R. Berger, E. Delamarche, H.P. Lang, C. Gerber, J.K. Gimzewski, E. Meyer and H.-J. Gntherodt, Surface stress in the self-assembly of alkanethiols on gold probed by a force microscopy technique,Appl. Phys. A, vol. 66, pp. S55S59, 1998.

Fig. 5. Surface stress changes when the MCs were exposed to the samples of 15mg/ml BSA and p53 antibody with concentration ranging from 20ng/ml to 20 g/ml. The MCs were stabilized before the injections at around 100s. The turbulence signals caused by injections come back within 10s.

IV.

CONCLUSION

Detection of p53 antibody is experimentally investigated using microcantilever biosensor with piezoresistive readout. Single crystalline silicon piezoresistors are fabricated using SOI wafers to improve the detection sensitivity. p53 antigen is covalently immobilized on the microcantilevers as the recognition molecules via 11-MUA SAM, and detection of p53 antibody is studied in detail. The experiment results show that specific and quantitative detection of p53 antibody ranging from 20ng/ml to 20g/ml is achieved, and the detection limit is 20ng/ml. Although the clinical detection limit of p53 antibody is below several ng/ml, these results demonstrate that after further improvement in sensor design and fabrication, the microcantilever biosensor has the potential

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