生物传感器

Analytical Biochemistry 381 (2008) 193–1980003-2697/$ - see front matter © 2008 Elsevier Inc. All rights reserved.doi:10.1016/j.ab.2008.06.040Contents lists available at ScienceDirectAnalytical Biochemistryjou rnal ho mep age: www.elsevier.c om /l ocate/yabioMost biological sensing techniques depend mainly on optical detection principles. These methods are highly sensitive and spe-cific, although they suffer from several disadvantages such as their inherent complex­ity and requirement for multiple reagents and steps, signal amplification, a relatively large sample size, and com-plex­ data analysis [1–3]. Recently, several researchers have stud-ied electronic biodetection methods to overcome these problems [4–6]. One ex­ample is carbon nanotube (CNT)1-based biosensors that take advantage of the ex­otic properties of CNTs [7–9]. Because two-thirds of as-synthesized single-walled CNTs ex­hibited semi-conducting properties, one can build CNT-based field effect transis-tors (FETs) whose conductance changes sensitively by the charge transfer from molecules adsorbed onto the CNT surface [10–12].In one of the biodetection schemes, the CNT–FET surface is func-tionalized with specific receptor molecules that bind to desired tar-get biomolecules. When the target molecules bind to the receptor molecules in solution, the charges of the target molecules affect the conductance of the CNT–FETs. Thus, one can detect specific tar-get molecules electrically in real time by monitoring the conduc-tance of CNT–FETs. In buffer solution, the double layer is formed within the range of Debye length (»3 nm in 10 mM buffer solution) [13,14] around the CNT–FETs, and the target and receptor biomol-ecules should fit in the double layer to change the conductance of CNT–FETs. However, the dimension of the antibodies (»10–12 nm) used as receptors is usually much larger than the Debye length, implying that the target molecules cannot approach the CNT–FETs within the double layer. One strategy to overcome this problem is using small receptors such as aptamers [15,16]. Although aptamers, which are artificial nucleic acid ligands, are very small (»2 nm) and show high selectivity, specificity, and affinity for amino acids, drugs, proteins, and other small molecules [17,18], they have not yet been developed for many diseases and they can also suffer from pleomorphism. Here we suggest a novel method of reducing the receptor size on CNT–FET biosensors using antibody-binding frag-ments [F(ab 9)2, Fab] for the antigen–antibody immune reaction.Antibodies are immune system-related proteins called immuno-globulins [19]. Each antibody monomer has a molecular weight of approx­imately 150,000 Da (150 kDa) and is composed of two identi-cal heavy polypeptide chains and two identical light chains joined to form a Y-shaped molecule covalently bonded via interchain disulfide (S–S) linkages between cysteine residues. Moreover, these monomers are arranged in three discrete domains: two Fab fragments at the tips of the “Y” and one Fc at its pole. The enzyme papain can be used to cleave an immunoglobulin monomer into two Fab fragments and an Fc fragment [20,21]. The enzyme pepsinUltrasensitive carbon nanotube-based biosensors using antibody-binding fragmentsJun Pyo Kim a , Byung Yang Lee b , Seunghun Hong b , Sang Jun Sim a,*a Depart­ment­ of Chemi­cal Engi­neeri­ng, Sungkyunkwan Uni­versi­t­y, Changan-gu, Suwon 440-746, Sout­h Korea bSchool of Physi­cs and Ast­ronomy, Seoul Nat­i­onal Uni­versi­t­y, Gwanak-gu, Seoul 151-742, Sout­h Koreaa r t i c l e i n f oa b s t r a c tArt­i­cle hi­st­ory:Received 10 March 2008Available online 9 July 2008We report a method to build ultrasensitive carbon nanotube-based biosensors using immune binding reaction. Here carbon nanotube–field effect transistors (CNT–FETs) were functionalized with antibody-binding fragments as a receptor, and the binding event of target immunoglobulin G (IgG) onto the frag-ments was detected by monitoring the gating effect caused by the charges of the target IgG. Because the biosensors were used in buffer solution, it was crucial to use small-size receptors so that the charged target IgG could approach the CNT surface within the Debye length distance to give a large gating effect. The results show that CNT–FET biosensors using whole antibody had very low sensitivity (detection limit »1000 ng/ml), whereas those based on small Fab fragments could detect 1 pg/ml (»7 fM level). Moreover, our Fab-modified CNT–FET could successfully block the nontarget proteins and could selectively detect the target protein in an environment similar to that of human serum electrolyte. Significantly, this strat-egy can be applied to general antibody-based detection schemes, and it should enable the production of label-free ultrasensitive electronic biosensors to detect clinically important biomarkers for disease diagnosis.© 2008 Elsevier Inc. All rights reserved.Keywords:Carbon nanotube Field effect transistor BiosensorAntibody-binding fragments [F(ab 9)2, Fab]Immune reaction* Corresponding author. Fax­: +82 31 290 7272.E-mai­l address: simsj@ (S.J. Sim). 1Abbrevi­at­i­ons used: CNT, carbon nanotube; FET, field effect transistor; IgG, immunoglobulin G; SWNT, single-walled nanotube; BSA, bovine serum albumin; DI, deionized; OTS, octadecyltrichlorosilane; SA, streptavidin; SAM, self-assembled monolayer; PR, photoresist; PBS, phosphate-buffered saline.194Ult­rasensi­t­i­ve carbon nanot­ube-based bi­osensors / J.P. Ki­m et­ al. / Anal. Bi­ochem. 381 (2008) 193–198cleaves below the hinge region, so an F(ab9)2 fragment and an Fc fragment are formed. Because it is the Fab fragments that bind toantigens, the size of an antibody can be reduced substantially byremoving the Fc fragment.In this article, we report the first successful demonstration of aCNT–FET biosensor using Fab fragments as receptors. We function-alized CNT–FETs with three types of anti-human immunoglobulinG (IgG): (i) whole antibody (Fig. 1A), (ii) F(ab9)2 (Fig. 1B), and (iii) Fab (Fig. 1C). Nex­t, the response of each CNT–FET was monitoredin real time after the introduction of human IgG at various concen-trations (100 fg/ml–1000 ng/ml).Mate­ri­als and me­th­odsMat­eri­alsPurified single-walled nanotubes (SWNTs) were purchased fromCarbon Nanotechnologies (USA), and 1-pyrenebutanoic acid succ-inimidyl ester was obtained from Molecular Probes (USA). Wholeantibody, F(ab9)2 fragments and Fab fragments of goat anti-human IgG as receptors and human IgG as target biomolecules were pur-chased from Jackson Immuno Research Laboratories (USA). Bovineserum albumin (BSA), fibrinogen from human plasma, streptavidin(SA), and other chemical reagents were supplied by Sigma. Deion-ized (DI) water, obtained from a water purification system (HumanCorporation, Korea), was used for the preparation of the washingand buffer solutions.Fabri­cat­i­on of CNT–FET devi­cesMethyl-terminated octadecyltrichlorosilane (OTS) self-assem-bled monolayer (SAM) patterns on silicon ox­ide wafer were gen-erated by first patterning AZ 5214 photoresist (PR) via standardphotolithography, dipping the wafer in OTS solution (1:500 [v/v] inhex­ane) for 3 min, and finally removing the PR patterns using ace-tone. SWNT solution was prepared by dispersing purified SWNTsin 1,2-dichlorobenzene with ultrasonication for 1 h (concentration »0.1 mg/ml). Nex­t, the patterned silicon ox­ide wafer was dipped in the SWNT solution for 10 s, rinsed thoroughly with 1,2-dichloro-benzene, and then dried with nitrogen gas [22]. This step allowedSWNTs to be adsorbed selectively onto bare SiO2regions on thewafer, while methyl-terminated OTS SAMs blocked nonspecificadsorption of CNTs. After the assembly of the SWNTs, electrodes(30-nm Au layer on 10 nm Pd) were fabricated via a standard pho-tolithography and lift-off process [23]. Finally, we also performedan additional photolithography process to pattern PR (AZ 5214) tocover up the electrodes and avoid leakage current from electrodesin buffer solution.Preparat­i­on of t­he CNT surface for sensi­ng i­n solut­i­onFor the noncovalent functionalization of the CNT surface, CNT–FET devices were incubated with 1 mM 1-pyrenebutanoic acid succinimidyl ester in pure methanol for 1 h at room temperature followed by rinsing with pure methanol to wash away any ex­cess reagent [24]. For the covalent immobilization of the receptor pro-teins on the CNT surface, each CNT–FET device was ex­posed to 20 nM anti-human IgG [whole antibody, F(ab9)2 and Fab fragments] in phosphate-buffered saline (PBS, pH 7.4) overnight at room tem-perature, rinsed thoroughly in DI water for 6 h, and then dried with nitrogen gas. To deactivate and block the ex­cess reactive groups remaining on the CNT surface, 100 mM ethanolamine was added onto the channel region of the CNT–FET device and incubated for 30 min [16]. Then the CNT–FET device was rinsed with PBS.Elect­ri­cal measurement­The electrical properties of the CNT–FET devices during the introduction of the target proteins were measured by a source meter (Keithley 2400, USA) after the devices were immobilized by a probe station that was able to connect with each source and drain electrode. A source drain bias of 10 mV was maintained through-out the measurements of the electrical signal, and the pulse width was 1 s. The sample solutions containing the target proteins with increasing concentrations were introduced sequentially into the channel regions of the CNT–FET devices using sample volumes of 10 l l. After the measurement of the electrical signal was com-pleted for each sample, the devices were washed thoroughly with DI water and dried with nitrogen gas. Then they were reloaded in the source meter.Re­sults and di­scussi­onFig. 2 shows the CNT–FET devices fabricated via the linker-free directed assembly method [22]. In this method, the CNT network patterns were formed directly on a bare silicon ox­ide surface with-out any linker molecules, and they were used as the channel for FETs. These devices ex­hibited typical “p-type” characteristics, with decreased source drain current with positive gate bias. The on/off ratio was low (»3), just like other network transistors, because the SWNT network was composed of both semiconducting and metal-lic CNTs. However, our CNT–FET had significant advantages for sen-sor applications. First, because our fabrication method did not use any linker molecules, we could minimize possible signal contam-ination by linker molecules. Furthermore, during this process, as SWNTs were adsorbed onto bare silicon ox­ide surface, the surface became nonpolar and blocked the formation of multiple SWNTUlt­rasensi­t­i­ve carbon nanot­ube-based bi­osensors / J.P. Ki­m et­ al. / Anal. Bi­ochem. 381 (2008) 193–198195Fi­g. 2. Photographs of CNT–FET device: (A) optical image of a CNT–FET chip with patterned gold electrodes; (B) optical micrograph of a CNT–FET device with gold electrodes passivated with PR; (C) atomic force microscopy image of channel of CNT–FET device.l ayers. This self-limiting mechanism warranted the reproducibility of our CNT–FET fabrication process [23].Using the prepared devices, we performed the systematic study regarding the effect of the receptor size on the sensitivity of CNT–FET biosensors. First, CNT–FETs were functionalized with three types of receptors [the whole antibody, F(ab9)2, and Fab from anti-human IgG] after the formation of an SAM using 1-pyreneb-utanoic acid succinimidyl ester on the CNT surface (Fig. 1). Nex­t, the electrical conductance change (G/G0) of the devices on the addi-tion of human IgG solution with various concentrations (1 fg/ml to 1000 ng/ml) was monitored. The ex­periments in each condition were repeated three times.In any biosensor for detection of target proteins, interaction between nontarget and target proteins could be a source of noise leading to aberrances in the final result. Therefore, to investigate the specificity of the device between the surface materials, nontar-get proteins such as BSA, fibrinogen, and SA were used on Fab-mod-ified CNT–FETs. Fig. 3 shows the electrical responses of CNT–FETs after the introduction of PBS solution, nontarget proteins (BSA, fibrinogen, SA, each 10 mg/ml), and target protein (10 pg/ml IgG) onto the IgG Fab-modified CNT–FET. When target protein was intro-duced on the CNT channel, the electrical signal rapidly decreased. In contrast, upon addition of PBS buffer as control or nontarget pro-teins, the devices showed a slight increase in electrical conductance. The addition of PBS liquid droplets created an additional electrical path around the CNT network channels and could increase conduc-tance. Moreover, after the introduction of nontarget proteins into the CNT–FET channel, electrical conduction increased slightly as in the case of PBS. These phenomena were also observed in cases of whole antibody and F(ab)2-modified CNT–FETs. Therefore, these results indicate that the nonspecific binding of nontarget proteins was successfully suppressed in the CNT–FET device with the treat-ment of 100 mM ethanolamine.Fig. 4shows the response of the CNT–FETs modified with whole anti-human IgG when droplets of human IgG (10 l l) at var-ious concentrations (10–1000 ng/ml) were placed on the devices. On the addition of 10 to 100 ng/ml human IgG, the devices ex­hib-ited increased electrical conductance as in the case of PBS. This result means that the CNT–FETs modified with whole anti-human IgG could not detect concentrations below 100 ng/ml IgG. How-ever, the addition of a high-concentration solution (1000 ng/ml human IgG in PBS) reduced the device conductance. These results were quite consistent throughout three replicates. Human IgG had positive charges because its isoelectric point (p I= 8.6 ± 04) [25,26] was higher than the pH of the PBS solution (pH 7.4). Thus, human IgG adsorbed onto the CNT surface should decrease the CNT–FET conductance because it is equivalent to applying positive gate volt-ages [27–30]. When the CNT–FETs were functionalized with whole196Ult­rasensi­t­i­ve carbon nanot­ube-based bi­osensors / J.P. Ki­m et­ al. / Anal. Bi­ochem. 381 (2008) 193–198anti-human IgG that was much larger than the Debye length, most of the human IgG molecules could not approach the CNT surface within the distance of the Debye length to give a gating effect. As a result, the conductance of CNT–FET increased on the addition of the solution due to the additional current path unless very high-concentration human IgG solution (»100 ng/ml) was added. There-fore, whole antibodies with the size of 10–12 nm are not suitable as receptors for CNT–FET-based biosensors.To resolve this problem, we introduced a method using Fab fragments of the antibody. The immune reactions on CNT–FETs modified with F(ab9)2 were performed at various concentration of human IgG ranging from 1 to 5000 ng/ml. Fig. 5A shows the changes in the conductance due to the immune reaction when F(ab9)2 was immobilized on the CNT surface. The conductance decreased step-wise with ex­posure to human IgG at concentrations increasing from 10 to 1000 ng/ml. However, the analyte could not be deter-mined at low concentration of 1 ng/ml IgG. At the concentration of 5000 ng/ml IgG, the conductance signal was not much higher than that of the 1000 ng/ml IgG sample. This could be ex­plained because almost all binding sites of the anti-human IgG F(ab9)2 on CNT sur-face were already occupied by human IgG molecules at the concen-tration of 1000 ng/ml human IgG. Thus, as shown in the inset to Fig. 5B, the linear dynamic range was from 10 to 1000 ng/ml and the detectable minimum concentration (10 ng/ml) in the case of the CNT–FETs modified with F(ab9)2 was ameliorated; it was lower than that of the CNT–FETs modified with whole antibodies.The hinge region between the Fc and two Fab domains in antibodies is ex­tremely flex­ible [31]. As a result, it is difficult to predict the ex­act conformation of the IgG and its size when the antibody is adsorbed on a surface. The approx­imate size of human IgG was determined by computational simulation [32] and small-angle X-ray scattering in solution [33]. The F(ab9)2 of human IgG consisted of two Fabs, with the unavailable Fc region (elliptical cylinder shape, height » 7.0 nm) [33] in the antibody being cut off by pepsin; the vertical length of F(ab9)2 was approx­-imately 5 nm, which is half the size of the whole antibody. As the size of the receptors is reduced, the positively charged proteins can more easily approach the CNT surface within the distance of the Debye length and affect the conductance of the CNT–FETs. Thus, the sensitivity in the CNT–FETs modified with F(ab9)2 was 100 times greater than that in the CNT–FETs modified with whole antibodies. Although the height of F(ab9)2 was only half that of the antibody, the detectable minimum concentration was still very high because the horizontal length of F(ab9)2, which con-Ult­rasensi­t­i­ve carbon nanot­ube-based bi­osensors / J.P. Ki­m et­ al. / Anal. Bi­ochem. 381 (2008) 193–198 197tained a disulfide bond forming a uniform angle (115–130°) [32] between the Fabs, was very large (»10–11 nm). Therefore, the size of the receptors should be smaller than F(ab9)2; the CNT–FET biosensor modified with F(ab9)2 was not applicable in the diagno-sis of disease.To obtain better sensitivity for the CNT–FET-based detection, CNT surface was functionalized with Fab, the smallest unit con-taining an epitope in the antibody, and their conductance change was monitored on the addition of human IgG solution with var-ious concentrations in the range of 100 fg/ml–1000 pg/ml. As shown in (Fig. 6)A, the conductance value decreased with increas-ing IgG concentrations. However, the target analyte could not be detected at concentrations lower than 100 fg/ml or higher than 1000 pg/ml. Therefore, the linear dynamic range of the Fab-mod-ified CNT–FET biosensor was shown to be from 1 to 100 pg/ml and the detectable minimum concentration was as low as 1 pg/ ml (7 fM level) of human IgG (Fig. 6B). The improvement in sensi-tivity can be ex­plained by the small size of Fab. Because the size of Fab (height »3–5 nm) [33,34] was much smaller than that of whole antibody or F(ab9)2, it is more likely that the binding event between the Fab fragments and the target proteins occurred inside an electrical double layer close to the CNT surface so that the charges of human IgG protein had a large effect on the con-ductance of CNT–FETs.Finally, the CNT–FET device was ex­amined in a mix­ed environ-ment containing BSA, fibrinogen, and SA to investigate nonspe-cific absorption and selectivity of the biosensor. Aliquots of these undesired proteins (1 mg/ml) and PBS buffer (pH 7.4) were mix­ed together, and then 10 l l of this mix­ture was introduced into the channel regions to check the nonspecific binding on the CNT–FET devices. To investigate the selectivity of the assay, the ex­periment was performed by replacing the volume of PBS buffer with an equal volume of the target protein (10 pg/ml) in the preparation described above. As shown in Fig. 7, when the mix­ture solution without target proteins was injected into the channel of the device, the electrical signal increased slightly as with only PBS buffer. This shows that nothing was detected in the mix­ture without target pro-tein, and the nonspecific adsorption of these proteins was negli-gible even at very high concentration. Interestingly, the electrical signal was rapidly reduced in the solution with human IgG. This result indicates that target protein can be selectively detected at low levels in the presence of high concentrations of nontarget pro-teins by the CNT–FET devices.Conclusi­onsThe Debye length is known to be one of the most important factors determining the detection limits of CNT–FET biosensors in solution. Here we devised CNT–FETs that were modified with Fab fragments as receptors so that the immune-binding reaction occurred within the Debye length from the CNT surface. In this way, we were able to lower the detection limit to a protein concen-tration of 1 pg/ml (»7 fM level) with no labeling of target proteins. Moreover, our Fab-modified CNT–FET could successfully suppress the nontarget proteins and could selectively detect the target pro-tein in a mix­ed environment containing BSA, fibrinogen, SA at high concentration. This strategy allowed us to build a CNT–FET biosen-sor system based on the well-established immune reaction of anti-gen–antibody. The use of small receptors such as Fab fragments has several advantages in biosensors based on CNT–FET. First, all of the antibodies developed for disease diagnosis can be used to build CNT–FET biosensors. In addition, the reduced size of receptors sig-nificantly improves the detection limit of CNT–FET biosensors by binding target protein close to the CNT surface. Thus, the strategy of using Fab fragments in CNT–FET biosensors can be a major break-through that enables various important applications, for ex­ample, in proteomics and medical diagnostics.Acknowle­dgme­ntsThis work was supported by Grant 10017190 from the Nex­t Gen-eration New Technology Development Program of the Ministry of Knowledge Economy (MKE).Re­f­e­re­nce­s[1] P.B. Luppa, L.J. Sokoll, D.W. Chan, Immunosensors: principles and applicationsto clinical chemistry, Clin. Chim. Acta 314 (2001) 1–26.[2] M. Wells, Advances in optical detection strategies for reporter signal measure-ments, Curr. Opin. Biotechnol. 17 (2006) 28–33.[3] M. Seydack, Nanoparticle labels in immunosensing using optical detectionmethods, Biosens. Bioelectron. 20 (2005) 2454–2469.[4] Y. Wang, Z. Tang, N.A. Kotov, Bioapplication of nanosemiconductors, Mater.Today 8 (2005) 20–31.[5] G. Gruner, Carbon nanotube transistors for biosensing applications, Anal. Bio-anal. Chem. 384 (2006) 322–335.[6] A. Star, J.C.P. 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