Changes of mutagenicity and antimutagenicity of the lactic fermented soymilk after exposure to acid, bile salt and heating

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An ultra sensitive DNA detection by using gold

nanoparticle multilayer in nano-gap electrodes

Chien-Ying Tsai

a

, Tien-Li Chang

a

, Chun-Chi Chen

b

,

Fu-Hsiang Ko

b

, Ping-Hei Chen

a,*

a

Department of Mechanical Engineering, National Taiwan University, No. 1, Roosevelt Road Sec. 4, Taipei 10617, Taiwan

b

National Nano Device Laboratories, Hsinchu 300, Taiwan Available online 11 January 2005

Abstract

This paper reports an electrical DNA detection method that detects target DNA at concentrations as low as 1 fM by using self-assembly multilayer gold nanoparticle structure between nano-gap electrodes. The distance of the gap between the electrodes is 300 nm and the height of the electrodes is 65 nm. A multilayer gold nanoparticle structure can be built on thin ﬁlm of SiO2silicon wafer. Bifunctional organic molecules are used to build up gold nanoparticle

monolayer on the wafer substrate. After hybridization among target DNA, 50_{-end thiol-modiﬁed probe DNA and}

30_{-end thiol-modiﬁed capture DNA; a second layer of gold nanoparticle is built up through a self-assembly process}

between gold nanoparticles and thiol-modiﬁed end of probe DNA. When the applied voltage is the same, electrical cur-rent through multilayer gold nanoparticle structure is much greater than that through monolayered gold nanoparticle structure. Therefore, target DNA in sample solution can be detected through a signiﬁcant degradation in electrical resis-tance from monolayered gold nanoparticle to multilayer gold nanoparticle structures. The concentration of target DNA in tested sample solutions ranges from 1 fM to 100 pM. The linear I–V curves of multilayer gold nanoparticle structures indicate the device proposed in this study can detect target DNA concentrations as low as 1 fM. Additional approaches are also suggested in this study to improve the sensitivity in DNA detection by using I–V measurements through mul-tilayer gold nanoparticle structures.

Keywords: DNA detection; Self-assembly; Gold nanoparticles; Bifunctional organic molecules; Monolayer; Multilayer

1. Introduction

In recent years, many approaches have been developed to detect DNA for diagnosis of genetic diseases. The success of these approaches heavily

*

Corresponding author. Tel./fax: +88 622 367 0781; fax: +88 622 363 1755.

E-mail address:phchen@ntu.edu.tw(P.-H. Chen).

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relies on the integration of interdisciplinary knowl-edge of molecular biology, chemistry and physics. Among these detection approaches, gold nanopar-ticles (AuNPs) that serve as tag material in both optical and electrical approaches reveal high sensi-tivity and selecsensi-tivity [1–6]. In our prior studies

[7,8], gold nanoparticles served as tag material for electrical detection of DNA hybridization. First, self-assembly gold nanoparticle monolayer is immobilized on SiO2 surface between nanogap electrodes by using a chemical compound, with 3-Mercaptopropyltrimethoxysilane (MPTMS). Then, gold nanoparticles could be added to the surface of the monolayer through hybridization among thiol-modiﬁed capture, thiol-modiﬁed probe, and target DNA single strands. A sharp rise in electric current through nanogap electrodes can be observed if additional layer of gold nanoparti-cles through DNA hybridization is formed on the self-assembly gold nanoparticle monolayer

[7]. In addition, the electric conductivity of self-assembly gold nanoparticle multilayer increases with the concentration of target DNA[7–9].

Metallic nanoparticles have been used to estab-lish self-assembly nanostructure of which physical and chemical properties have been investigated in recent years. In particular, gold nanoparticles can be easily prepared and have the characteristic of biocompatibility [10]. Some new devices have been developed for the application of immunoas-say by making use of the novel properties of nanostructure that is self-assembled by gold nanoparticles [11]. However, the properties of gold nanostructure can vary signiﬁcantly with the size of gold nanoparticles and the pitch be-tween gold nanoparticles in the nanostructures

[10–13]. A gold-ampliﬁed sandwich immunoassay for the detection of human immunoglobulin G has also been developed by Natan and co-workers [13]. The sensitivity of their immunoas-say can reach 1 pM. They have also conducted a series of studies on the eﬀect of particle size and surface coverage on the detective sensitivity of immunoassay [14,15]. In 2002, Mirkin and his group immobilized AuNPs on the gap surface between two 20-micro-gap electrodes through hybridization among target DNA (tDNA), cap-ture DNA (cDNA), and probe DNA (pDNA)

[16]. However, the current through the monolayer AuNPs is too small to detect. In order to enhance the current signal, an approach with a silver en-hancer solution of AgNO3 and hydroquinone was proposed in their study.

In this study, an electrical approach is used to detect DNA hybridization through the establish-ment of gold nanostructure on the gap surface be-tween two electrodes. The gold nanostructure is established by such a way that employs functional single strand DNA as linking chemical. Electrical detection for DNA hybridization has some advan-tages over the optical approach in rapid clinical diagnosis of genetic diseases. These advantages in-clude easier operation, less instrumental cost, and denser measuring points. However, the sensitivity and consistency of electrical detection should be improved signiﬁcantly in order to have practical application.

In this study, we describe the use of bioconju-gate that consists of oligonucleotide probes and colloidal AuNPs for DNA hybridization detection via the construction of multilayer nanostructure on top of the bottom layer through a layer-by-layer process. Specifically, oligonucleotide probes are conjugated to colloidal AuNPs. They are used to selectively recognize surface-confined target DNA via sequence-specific hybridization with in situ detection. The substrate between the nano-gap electrodes, a chemical compound 3-aminopropyltrimethoxysilane (APTMS), is used to modify the substrate surface. One end of the APTMS compound is to silanize the substrate sur-face while the thiol end of the APTMS compound is used to bind the gold nanoparticles. At high concentration of target DNA, gold nanoparticle multilayer is then established through DNA hybridization among thiol-modified capture, thiol-modified probe, and target DNA single strands. At low concentration of target DNA, it is required to establish a middle layer for enhanc-ing electrical signal through electrodes. A substan-tial improvement in electrical response is achieved compared with an unamplified detection situation. To further support the validity of this approach, either salt solution or a thermal denaturation pro-cess can be used to prove whether DNA hybridiza-tion is complementary.

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In addition, this study aims at developing an electrical detection method for DNA hybridization without any ampliﬁcation of target DNA through a polymerase chain reaction that is usually time consuming. Therefore, the detection sensitivity of this approach must reach 1 fM.

2. Experimental details

2.1. Preparation of gold nanoparticles

In this study, the first step is to prepare dis-persed gold nanoparticles in aqueous solution by using a chemical reduction method[16]. In a flask, 80 ml distill water and 1 mg HAuCl4 are mixed, and then it was heated up to 60°C with vigorous stirring. An addition of 20 ml water solution with 0.05 mg trisodium citrate and 0.01 mg tannic acid is added into the flask. The color of the solution

gradually changes to crimson and then violet around 3 min after the solution starts to change color. Afterwards, the aqueous solution is heated to boiling for an additional 15 min. After returning to room temperature, colloidal gold nanoparticles are formed in the solution.

The solution of AuNPs is measured by a Hitachi U3310 UV–Vis spectrometer and a high-resolution transmission electronic micrograph (HR-TEM, model: H-7000, Hitachi) to determine the size of gold nanoparticles. The HR-TEM micrograph and UV–Vis absorption spectrum of gold nanoparticles are shown in Fig. 1(a) and (b), respectively. The UV–Vis absorption spectrum of AuNPs solution shows a strong surface plasma resonance at a wavelength of 523 nm. From the HR-TEM micrograph of gold nanoparticles, the diameter of monodispersed Au colloidal is found to be in the range of 12 ± 4 nm. It is worth noting that the size of gold nanoparticles in the solution

Fig. 1. Measured results of gold nanoparticles with an average diameter of 12 nm: (a) TEM micrograph; (b) UV–Vis absorption spectrum.

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can be varied by changing the concentration of tri-sodium citrate in the aqueous solution.

2.2. The fabrication of nano-gap gold electrodes The procedure for fabricating nano-gap elec-trode is shown inFig. 2. The procedure for estab-lishing the multilayer structure of gold nanoparticles between electrodes is illustrated in

Fig. 3. The ﬁrst step for manufacturing nano-gap electrode is to sandwich a p-type Si(1 0 0) wafer (phoenix, San Jose, CA) with a 2000 A˚ SiO2thin ﬁlm using PECVD. It is then followed by spin coating of a 7000 A˚ thick photoresistant on the SiO2 surface. The pattern of electrodes on the photoresistant is obtained by using a direct writing approach with an E-beam writer. After the wafer is exposed to the E-beam writer, it is placed into

developed etchant to yield a 300 nm-gap on the photoresistant. The next step is to deposit titanium of 5 nm thick by using a thermal deposition pro-cess. It is then followed by a deposition of 35 nm gold thin ﬁlm. The ﬁnal step is to immerse the sil-icon wafer in acetone solution for 2 h. The elec-trodes with 300–600 nm nanogap can be obtained on the silicon wafer.

2.3. Establishing self-assembly gold nanoparticle monolayer and multilayer

To clean thoroughly, the substrate is rinsed with water in an ultrasonic cleaner for 5 min. It is then immersed in the 1 mM 3-aminopropyltri-methoxysilane (Sigma Chemical Co. St. louis, MO) of DMSO solution for at least 2 h at room temperature, followed by rinsing with DMSO

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and dried under N2. The functionalized substrate is immersed in the AuNPs solution overnight, then rinsed with distilled water and dried under N2.

The sequences of single strand capture, target, and probe DNAs as well as single base-pair mis-match of target oligonucleotides (cDNA, tDNA, pDNA and single-bp mismatch tDNA) are shown in Fig. 4. The oligonucleotides are prepared according to standard protocols[16].

The procedure to establish double layer of gold nanoparticles can be shown in Fig. 5. Except the middle layers, both bottom and top layers are established by using the same chemical compounds for the different procedures, APTMS for the bot-tom layer and DNA hybridization for the top layer. The middle layer is formed by using hexan-edithiol as a linking chemical compound. It is worth noting that the number of middle layer can be greater than one. APTMS, whose thiol end is bound with gold nanoparticle, modifies sili-con oxide substrate surface. To perform the detec-tion of target DNA, cDNA (100 lL, 1 lM) is deaerated in 10 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, J.T. Baker Chem. Co.), 5 mM EDTA buffer, pH 6.6 (HEPES buffer). The self-assembled gold-particle monolayer sub-strate is immersed in the cDNA solution for 15 h at room temperature, followed by rinsing with 50 mM sodium phosphate, 1 M NaCl, pH 6.5 (SPSC buffer) to remove non-covalently bound DNA, and is dried under N2. The substrate is im-mersed in four different concentrations of tDNA (0.1 lM, 1 nM, 10 pM and 1 fM, 24-mer) solution (100 lL) and pDNA solution (12-mer, 100 lL, 0.1 lM) for 2 h to hybridize, followed by immers-ing in a SPSC buffer to remove excess reagents. The substrate is then immersed in a solution of AuNPs in 0.3 M PBS buffer (0.3 M NaCl,

Fig. 3. The SEM images of nano-gap electrode with a magniﬁcation of (a) 300, and (b) 4000. The electrodes include gate, drain and source (thickness, 350 A˚ ; the distance of gap, 300 nm).

5'- GGA TTA TTG TTA AAT ATT GAT AAG GAT-3'

captureDNA

targetDNA

3'-HS-A -CCT AAT AAC AAT-5'

probeDNA 3'-TTA TAA CTA TTC CTA-A -SH-5'

10

single base pair mismatch of targetDNA

5'- GGA TTA TCG TTA AAT ATT GAT AAG GAT-3'

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10 mM NaH2PO4/Na2HPO4, pH 7), followed by washing with the 0.3 M PBS buﬀer to remove ex-cess AuNPs and is dried under N2. If hybridization among DNAs (pDNA, cDNA/complementary tDNA or single-bp mismatch tDNA) does occur, the top layer of gold nanoparticles is established between nano-gap electrodes. If there is no hybrid-ization, only monolayer of gold nanoparticles is established. However, a higher electric current can be detected through self-assembly multilayer gold nanoparticles if target DNA, probe DNA, and nanoparticles are placed in the solution at the same time. When gold nanoparticles are placed in the solution after the hybridization between tar-get, probe, and capture DNAs, the electric current is not as high. In this study, the latter approach for establishing self-assembly gold nanoparticle mutli-layer is used.

For verifying the speciﬁcity of target DNA, it is required to check whether the hybridization among DNAs has any mis-match. To check it, the chip can be washed with a 0.01 M PBS (0.01 M NaCl, 10 mM NaH2PO4/Na2HPO4, pH 7) buﬀer at room temperature[16]. If the hybrid-ization among DNAs is not fully complementary,

the top layer of gold nanoparticles is washed away. As a result, the electric current is reduced signiﬁ-cantly back to a value that is obtained for self-assembly gold nanoparticle monolayer. The I–V curves of self-assembly gold nanoparticle multi-layer are measured by a HP 4156A precision semi-conductor parameter analyzer. The applied

Fig. 5. Chemistry of the attachment of thiol-modiﬁed DNA oligomers to self-assembly AuNPs monolayer. (a) Surface modiﬁcation with THMS, (b) Addition of AuNPs reacts to the thiol molecule, (c) An alkanethiol-cDNA reacts with AuNPs monolayer, (d) tDNA added and hybridized with cDNA and pDNA, thiol reacts with multilayer AuNPs, (e) The thiol-DNA oligomer subsequently reacts with the AuNPs surface, to yield the self-assembly of multilayer of AuNPs on the below illustration.

Fig. 6. Current–voltage curve for the monolayer AuNPs at a scan rate of 10 mV/s.

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voltage ranges from1 to +1 V with a scan rate of 1 mV/s.

3. Results and discussion

Methods for electrical detection of DNA with AuNPs were previously reported by Tsai and co-workers [7,8]. However, the lowest concentration

of target DNA detected in their study is 10 pM. In this study, the gap distance between electrodes is reduced from 600 nm in their study to 300 nm.

Fig. 3 shows the FE-SEM micrographs of nano-gap electrodes. In this study, a ﬁeld-emission scan-ning electron microscopy (FE-SEM: JEOL, JSM-6500F) is used to observe the nano-gap elec-trodes and self-assembly gold nanoparticle multi-layer on the surface between electrodes. Once

Fig. 7. FE-SEM images of the AuNPs multilayer at diﬀerent concentrations of target DNA: (a) 1 fM, (c) 10 fM, and (e) 100 fM. Current–voltage curves of the AuNPs multilayer at diﬀerent concentrations of target DNA: (b) 1 fM, (d) 10 fM, and (f) 100 fM. Here, tDNAs were hybridized to cDNA and pDNA in 0.3 M PBS for 2 h in all experiments.

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self-assembly gold nanoparticle multilayer is estab-lished on the silicon wafer, the color of biochip at measuring spot turns into deep brown.

Current–voltage (I–V) behavior of the device is measured via ohmic contacts as shown in

Fig. 6. If there is no AuNPs in the nano gap, the current of the device would be lower than 50 fA as observed. Once the monolayer AuNPs is established on the gap surface between

elec-trodes, the current of the monolayer AuNPs is still lower than 1 pA. Our precious study [8] em-ployed MPTMS to immobilize gold nanoparti-cles to establish the monolayer AuNPs on SiO2 surface. Note that the particle density of the monolayer AuNPs increases from 1230 parti-cles/lm2 to 1450 particles/lm2

if the MPTMS used in the precious study for immobilizing gold nanoparticles is replaced by ATPMS.

Fig. 8. FE-SEM images of the AuNPs multilayer at diﬀerent concentrations of target DNA: (a) 1 pM, (c) 10 pM, and (e) 100 pM. Current–voltage curves of the AuNPs multilayer at diﬀerent concentrations of target DNA: (b) 1 pM, (d) 10 pM and (f) 100 pM. Here, tDNAs were hybridized to cDNA and pDNA in 0.3 M PBS for 2 h in all experiments.

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Fig. 7 shows the FE-SEM images and the I–V curves of the multilayer AuNPs at different tDNA concentrations of 1, 10 and 100 fM.Fig. 8 shows the FE-SEM images and the I–V curves of the multilayer AuNPs at different tDNA concentra-tions of 1, 10 and 100 pM. Electrons tunnel more readily through the junction when enough energy is supplied. The linear curve is typical of ohmic de-vices. Figs. 7 and 8 show the FE-SEM images of the SiO2surface treated in a manner identical to the treatment of the electrode gaps before and after incorporation of the second AuNPs via DNA immobilization, which clearly indicates that AuNPs density increases with increasing tDNA concentration. Higher tDNA concentration in-creases the AuNPs density on SiO2 surface. The average densities of the particles are 1500, 1550, 1650, 1900, 2030, 2200 particles/ lm2as shown in Figs. 7(a), (c), (e) and 8(g), (i), (k). However, in the absence of tDNA and after the chip is washed with a 0.3 M PBS buffer, the same signal as to the monolayer of AuNPs is ob-served, less than 1 pA with an applied voltage ranging from 1 to 1 V. Using this method, we can target oligonucleotide over a wide concentra-tion range. The right side of Fig. 7 and 8 show the I–V curves of the nano-gap electrode without the silver enhancing approach [16]. Using this method, electrical detection is feasible in the 1 fM to 100 pM concentration range.Fig. 9shows a dependence of electric resistance of the multi-layer AuNPs with the target DNA concentration (M). It can be estimated that the correlation be-tween the target DNA concentration/tDNA/versus resistance/R/of the multilayer AuNPs is

logð=tDNA=Þ ¼ 4:0625 1:0375 log =R=: The difference in conductivity of multilayer with different concentration of tDNA can be readily ob-served at room temperature. The self-assembly procedure described above provides a versatile and convenient method for DNA detection. The observation of increased AuNP density is consis-tent with our anticipation for DNA hybridization. The system is based on nano-gap electrodes, it is positioned for massive multiplexing through the use of electronic properties by using different com-pounds for the assembly of AuNPs multilayer.

4. Conclusion

An electrical detection on DNA hybridization with multilayer gold nanoparticles is described in this paper. The system is based on gold nanoparti-cles synthesized by a chemical reduction method and a monolayer of the particles established on the surface between electrodes. Significantly, the hybridization within DNA can then be identified from an increase in measured current through elec-trodes due to additional layers of gold nanoparti-cles, which are established through hybridization among DNAs. The tDNA concentration affects the measured magnitude of electric current through multilayer gold nanoparticles.

Acknowledgment

We deeply appreciate the ﬁnancial support by the Nation Science Council under the Grant num-ber of NSC-92-2212-E-002-050.

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