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Biosensors and Bioelectronics 24 (2009) 1241–1245

Contents lists available at ScienceDirect

Biosensors and Bioelectronics

journa l homepage: www.e lsev ier .com/ locate /b ios

abel-free DNA sensor based on organic thin film transistors

eng Yana,∗, Sheung Man Moka, Jinjiang Yub, Helen L.W. Chana, Mo Yangb

Department of Applied Physics, The Hong Kong Polytechnic University, Hong Kong, ChinaDepartment of Health Technology and Informatics, The Hong Kong Polytechnic University, Hong Kong, China

r t i c l e i n f o

rticle history:eceived 10 April 2008

a b s t r a c t

Organic thin film transistors (OTFTs) are excellent candidates for the application on disposable sensors dueto their potentially low-cost fabrication process. A novel DNA sensor based on OTFTs with semiconducting

eceived in revised form 1 July 2008ccepted 14 July 2008vailable online 26 July 2008

eywords:rganic thin film transistorrganic semiconductorNA sensor

polymer poly(3-hexylthiophene) has been fabricated by solution process. Both single- and double-strandDNA molecules are immobilized on the surface of the Au source/drain electrodes of different OTFT devices,producing a dramatic change in the performance of the devices, which is attributed to the increase ofthe contact resistances at the source/drain electrodes. Single-strand DNA and double-strand DNA aredifferentiated successfully in the experiments indicating that this is a promising technique for sensingDNA hybridization without labelling.

© 2008 Elsevier B.V. All rights reserved.

. Introduction

Detecting small quantities of biomolecules is paramount in theiagnosis of disease, drug discovery, and basic researches. Espe-ially, it is of great scientific and economic importance to developechniques for nucleic acid detection, which have broad potentialpplications including gene expression monitoring, pharmacoge-omic research and drug discovery, clinical diagnostics, viral andacterial identification, detection of biowarfare and bioterrorismgents, and forensic and genetic identification, etc. To exploit thesepportunities, DNA sensors are required to provide a combina-ion of high sensitivity, selectivity, speed, portability, and low cost.herefore, DNA microarray (DNA chip) technology has been devel-ped to offer an unprecedented simultaneous and multiplexednalysis in a high-throughput screening format.

Label-free techniques are of special interest since incorpora-ion of a labelling step into a nucleic acid assay makes it moreomplex, cumbersome and expensive. Recently, there has beennterest in various technologies such as electrochemical detectionBoon et al., 2002), surface vibration spectroscopy (Miyamoto etl., 2005), atomic force microscopy (AFM) (Wang and Bard, 2001),canning Kelvin probe microscopy (SKM) (Thompson et al., 2005),enetic field effect transistor (FET) (Estrela et al., 2005; Estrale andigliorato, 2007; Pouthas et al., 2004; Sakata et al., 2004; Zhang

nd Subramanian, 2007) and microcantilever (Shekhawat et al.,006; Wu et al., 2001) to realize high sensitive, label-free, DNAicroarrays. Compared with the other techniques, genetic FET has

∗ Corresponding author. Tel.: +852 2766 4054; fax: +852 2333 7629.E-mail address: apafyan@polyu.edu.hk (F. Yan).

the advantage of being able to be miniaturized without losing sig-nal to noise ratio since the channel current of a FET is proportionalto the width/length ratio of the channel and not related to the areaof the device (Sze, 1981). Therefore FET is ideal for the applica-tion in small-sized, high-density and multi-functional microarraysensors (Yan et al., 2005). On the other hand, biosensors based onFETs can be easily integrated with circuitry to form self-supportedDNA detection platform since FET is the key component of integratecircuit.

DNA sensors based on various FETs have been reported. An inte-grated array of silicon FET for electronic detection of label-free DNAhave developed (Pouthas et al., 2004), which show a shift of gatevoltage for about 80 meV corresponding to the adsorption of DNAto the gate insulator −SiO2. Sakata et al. (2004) have studied agenetic FET based on n-type silicon FET with Si3N4 gate insula-tor. Single-strand DNA probes are immobilized on the surface ofgate insulator Si3N4. Hybridization with target DNA on the sen-sor has induced a shift of threshold voltage for 11 meV. Estrela etal. (2005) have developed DNA sensors based on extended gatepolycrystalline silicon thin film transistors (TFT). A parallel shiftof transfer characteristics (drain current versus gate voltage) of355 meV has been observed after the hybridization of single-strandDNA (ssDNA) probes immobilized on an extended Au gate electrodewith its complementary strand. All of the aforementioned geneticFETs are based on electronic detection of intrinsic charge of DNAmolecules (negative charge) in an electrolyte. DNA molecules in theelectrolyte are screened by mobile counter ions and thus inducea surface potential drop that modulates gate voltage applied onthe transistor. The potential drop is influenced by various factorsincluding the concentration of ions in the electrolyte, DNA concen-tration in the surface and the length of DNA strand (Pouthas et al.,

956-5663/$ – see front matter © 2008 Elsevier B.V. All rights reserved.oi:10.1016/j.bios.2008.07.030

1242 F. Yan et al. / Biosensors and Bioelectronics 24 (2009) 1241–1245

2004). So variations of the threshold voltage of the TFT due to DNAhybridization are diverse in different reports.

Organic thin film transistors (OTFTs) are excellent candidatesfor use in disposable sensors for their easy and cheap fabricationas compared with their inorganic counterparts (Sirringhaus, 2005;Yan et al., 2007). Organic materials can be dissolved in varioussolvents, so that transistors can be coated or printed at low tem-perature. In addition, organic semiconductors are biocompatibleand flexible thus they can be integrated with biological systems(McQuade et al., 2000; Berggren and Richter-Dahlfors, 2007; Barticand Borghs, 2006). There are several types of OTFT-based chemicalsensors or biosensors that have been reported. Loi and Manunza(2005) have developed organic ion sensitive field effect transistor(ISFET) on flexible plastic film that acts both substrate and gatedielectric. Tanese et al. (2005) reported gas sensor based on OTFTswith a gas sensitive organic semiconductor layer.

Zhang and Subramanian (2007) reported a DNA sensor based onpentacene TFT, in which DNA molecules are immobilized on the sur-face of semiconductor layer and an unambiguous doping-inducedthreshold voltage shift up to 20 V has been observed. However, theDNA molecules need to be immobilized on the surface of pentacene,which may decrease the stability and repeatability of the devicesince pentacene film is sensitive to moisture and some ions. To ourbest knowledge, this is the only paper that reports DNA sensingperformance based on OTFTs. In this paper, we report another typeof DNA sensor based on OTFTs, which shows big change of device

performance when DNA molecules are immobilized and hybridizedon the surface of source/drain electrodes.

2. Experiments

2.1. Substrate preparation

As shown in Fig. 1, OTFTs have been fabricated on a Si sub-strate. Highly doped n-type silicon wafer with 500 nm thick SiO2was used as the starting substrate, in which the SiO2 film and n-type Si acted as the gate insulator and gate electrode, respectively.Then Au source/drain electrodes were deposited on top of the SiO2film through a shadow mask with thermal evaporation. The channellength and width for any device were 0.2 and 2 mm, respectively.

2.2. DNA immobilization and hybridization

To detect the effect of DNA immobilization and hybridization,three identical chips were used for one set of samples in theexperiment. The gold electrodes were first washed with acetone,and followed by deionized (DI) water and phosphate buffer solu-tion (PBS) (pH 7.4). Then, the probe single-strand DNA (21 bases,3′-/3ThioMC3-D/TTT TGT CCT TTG TCG ATA CTG-5′ (HPLC purifi-cation)) dissolved in phosphate buffer solution (PBS) (2.0 �M DNAin 75 �L PBS) was dropped on the two chips and left for a certainperiod of time (t) to immobilize ssDNA on the Au source/drain elec-

Fig. 1. Scheme showing steps to prepare OTFTs incorporated with DNA layer in the devices. The thickness of SiO2 is 500 nm. The channel width and length of the OTFTs are2 and 0.2 mm, respectively.

F. Yan et al. / Biosensors and Bioelec

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bifttrol sample. Fig. 3a shows channel current as a function of gatevoltage measured at a constant drain voltage (transfer characteris-tic, of the three samples (control, ssDNA and dsDNA). Fig. 3b showsthe channel current as a function of drain voltage VDS under severaldifferent gate voltages (output characteristics, VGS = −40, −30, −20,

Fig. 2. Florescence images of gold electrodes after the hybridization of DNA.

rodes. After the immobilization, the substrate was washed againith PBS. Target DNA (5′-/56-FAM/CAG GAA ACA GCT ATG AC-3′,

7 bases, HPLC purification) dissolved in PBS (2.0 �M DNA in 75 �LBS) was dropped on one of the two chips and left for around 2 h forybridization. After the interaction, the substrate was again washedith PBS.

Fluorescence images of labelled DNA were obtained from a flu-rescence microscope (Nikon ECLIPSE 80i, Japan). Since the targetNA was labelled with 56-FAM group, the immobilization of probe

sDNA and hybridization of the target DNA could be confirmednder the fluorescent microscopy, as shown in Fig. 2.

.3. Fabrication of organic thin film transistor

Then organic semiconductor thin films were spin-coated on topf the three chips after being washed with DI water. Here, the threehips include one control sample without DNA on Au electrodes,ne sample with ssDNA and one sample with dsDNA immobilizedn Au electrodes. We chose regio-regular poly(3-hexiothiophene)rr-P3HT) (regioregularity is ∼98.5%, from Aldrich) as the semi-onductor material since rr-P3HT has a relatively high-field effectobility (Sirringhaus et al., 1998), which is close to the value for

morphous Si. Thus, rr-P3HT is a promising material for the appli-ations in OTFT sensors. In the experiment, rr-P3HT was dissolvedn chloroform with the concentration of 10 mg/ml and spin-coatedn the samples with a film thickness of ∼20 nm. Here, the thicknessf the polymer film is not important for the sensor since OTFT is annterfacial device and the channel current normal passes throughvery thin layer (less than several nanometers) near the insulator

Sze, 1981). Then all of the samples were annealed at 60 ◦C for 1 ho remove solvent and improve the crystallinity of P3HT layer.

.4. Signal acquisition

The DNA sensors based on OTFTs have been characterized bysing Agilent 4156C in a glovebox filled with high-purity N2. Deviceerformances of OTFTs, including transfer characteristics and out-ut characteristics of each device, have been measured. For transferharacteristics, the channel current IDS between source and drainas measured as a function of gate voltage VG under a constantrain voltage VDS. For output characteristics, the channel current

DS was measured as a function of drain voltage VDS under a con-

tant gate voltage VG and different VG results in a different curvef IDS versus VDS. To obtain a stable result, channel current waseasured for 2 s for each value of applied voltage.One important parameter of OTFT is the field effect mobility of

arriers in the channel. In our experiment, the field effect mobility

FtVFt

tronics 24 (2009) 1241–1245 1243

f holes � in the device can be calculated from the saturation chan-el current of the device IS (|VDS| > |VG|) as a function of applied gateoltage VG:

= 2L

WCi

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ISdVG

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(1)

i = εiε0

ti

here Ci is the capacitance of gate dielectric per unit area; εi ishe relative dielectric constant of gate insulator; ε0 is the dielectriconstant in vacuum; ti is the thickness of the gate insulator; W andare channel width and length of the OTFT, respectively;

. Results and discussion

To find the influence of the density of DNA molecules immo-ilized on the Au electrode, several sets of samples with different

mmobilization time (t = 4, 8, 24 and 48 h) of probe ssDNA have beenabricated. For the set of samples with lower density of DNA (t = 4 h),he performance of OTFTs already exhibits big difference to the con-

ig. 3. Performance of the OTFTs with probe immobilized on Au source/drain elec-rodes for 4 h. (a) Transfer characteristics of three OTFTS (control, ssDNA and dsDNA):DS = −40 V; (b) output characteristics of three OTFTs (control, ssDNA and dsDNA).or each device (different colour corresponds to different device), from top to bot-om, the curves are measured at the VGS of −40, −30, −20, −10 and 0 V.

1244 F. Yan et al. / Biosensors and Bioelectronics 24 (2009) 1241–1245

Fig. 4. Performance of the OTFTs with probe immobilized on Au source/drain elec-tddt

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rodes for 48 h. (a) Transfer characteristics of three OTFTS (control, ssDNA andsDNA): VDS = −40 V; (b) output characteristics of three OTFTs (control, ssDNA andsDNA). For each device (different colour corresponds to different device), from topo bottom, the curves are measured at the VGS of −40, −30, −20, −10 and 0 V.

10, 0 V) of the three samples. At the same applied voltages, theontrol sample shows the maximum channel current and the sam-le with dsDNA layer shows the minimum. Therefore a DNA layeretween the metal electrode and semiconductor layer decreaseshe channel current and dsDNA shows bigger effect than ssDNA.

For the samples with the highest density of DNA (t = 48 h) immo-ilized on the electrodes, the transfer and output characteristicsave been measured at the same condition, as shown in Fig. 4a and, respectively. The device performance exhibits dramatic changeompared with the control sample. The on current of the deviceith ssDNA is much lower than that of the control sample andigher than that with dsDNA. The threshold voltage of the sampleith ssDNA is very different from the other two samples. We think

t is due to some ssDNA being immobilized on the channel regionesides on Au electrodes since the immobilization time is relatively

ong (48 h). Threshold voltage of an OTFT can shift to positive valuef there is some negative charge immobilized in the channel region.n this sample, the positive shift can be attributed to the intrinsicegative charge of DNA molecules in the channel region. This resultlso suggests that it is not necessary to immobilize DNA for too longime.

Field effect mobility of carriers in the OTFTs as given by Eq. (1)

s influenced by the DNA density. For each sample, we have fabri-ated three identical devices showing very similar performance. Ashown in Fig. 5, the field effect mobility for different immobiliza-ion time of DNA is the average value of the mobilities calculatedrom the saturation current of the three identical OTFTs. For each

saida

ig. 5. Field effect mobilities of the OTFTs with ssDNA and dsDNA layers immobilizedn Au source/drain electrodes as a function of immobilization time t (t = 4, 8, 24 and8 h) of DNA probe and t = 0 corresponds to the control sample.

evice, the variation of mobility is within ±10% of the average value.herefore the error bar we set is 10% of the results shown in Fig. 5. Itan be found that field effect mobility decreases with the increasef immobilization time of probe DNA on the Au electrodes. SinceNA is immobilized only on the source/drain electrodes, we con-

ider that a DNA layer increases the contact resistance of the OTFThus decreases the channel current.

The effect of DNA layer on the contact resistance can bettributed to two possible reasons. One is due to the high resistancef DNA layer connected in series with the channel resistance. Sincesingle-strand oligonucleotide has 0.34 nm distance between baseairs (Thompson et al., 2005), the maximum length of a DNA with1 bases is about 7 nm, which also can be regarded as the maxi-um thickness of the DNA layer. So the thickness of DNA layer is

ve orders of magnitude smaller than the channel length (0.2 mm).n addition, previous studies on single DNA ropes indicate that DNAehaves as a good semiconductor (Fink and Schonenberger, 1999;orath et al., 2000). Therefore the influence of the resistance of DNAayer on the channel current is negligible.

Another possible reason can be due to the decrease of injec-ion current from source electrode to the channel, which has beenegarded as the main reason for the contact effect in OTFTs (Burgit al., 2002; Hong et al., 2007). The contact effect at source/drains very important for the performance of a TFT, which is normallynduced by a higher (lower) Fermi level of the metal source/drainlectrode relative to the HOMO (LUMO) level of the semiconductorayer for p-channel (n-channel) devices. In other words, it is dueo a potential barrier at the source or drain contact. The contactesistance is limited by a tunnelling or hopping process of carrierscross the barrier. Therefore, a small change of the work functionf source/drain electrodes can dramatically influence the contactesistance and the channel current. For a p-channel device, theower the work function of the electrodes is the higher the contactesistance will be. The highest occupied molecular orbital (HOMO)evel of P3HT (−5.2 eV) (Zaumseil and Sirringhaus, 2007) is lowerhan the Fermi level of Au (−5.1 eV). Therefore any decrease of theurface potential of Au electrode can induce increase of contactesistance.

It has been reported that immobilization of probe ssDNAolecules on Au electrode and thereafter hybridization with com-

limentary strands may decrease the work function of the Auurface, as confirmed by SKM measurement (Thompson et al., 2005)

nd other experiments (Estrela et al., 2005). This effect is normallynduced by surface dipole formed by intrinsic charge of DNA. Soetecting a work function change induced by a biological inter-ction can be a technique employed in nucleic acid microarray

F. Yan et al. / Biosensors and Bioelectronics 24 (2009) 1241–1245 1245

technology. In our experiment, since the immobilized DNA layerdecreases the work function of the Au source/drain electrodes,increase of contact resistance at source/drain electrodes can beexpected. Therefore the decrease of the channel current and thefield effect mobility of the devices with DNA immobilized on Ausource/drain electrode can be attributed to the increase of the con-tact resistance and this effect is bigger for higher density of DNA.

We can find the decrease of the channel current due to theimmobilization of dsDNA is bigger than that of ssDNA, thus dsDNAcan change the work function of the electrode more dramati-cally. Therefore ssDNA and dsDNA are differentiated successfullyin the experiments indicating that this is a promising technique forsensing DNA hybridization without labelling. Since it is a maturetechnique to immobilize DNA molecules on Au electrodes, highdensity of TFTs functionalized with different DNA probes can befabricated on a single chip (Thompson et al., 2005). Compared withthe OTFT-based DNA sensor developed by Zhang and Subramanian(2007), our device can be immobilized with DNA more conve-niently. In addition, the organic semiconducting layer in our deviceis coated after the DNA immobilization, therefore unwanted dopingeffect to organic layer will not occur and a more stable perfor-mance can be expected in the device. We consider that this typeof OTFTs can be developed as low cost, label-free and disposableDNA sensing microarrays. Further work will be carried out to studythe effect of DNA sequence, length and mismatch of hybridizationon the device performance and the effect of using different typesof organic semiconductors as the active layer in an OTFT.

4. Conclusions

In conclusion, DNA immobilized on the surface of source/drainAu electrodes of OTFT can dramatically change the channel currentand the field effect mobility of the device, which is attributed tothe increase of contact resistance at the source/drain electrodes.The increase of contact resistance can be explained in terms of thedecrease of work function of the source/drain Au electrode afterDNA immobilization and hybridization. Based on this technique the

OTFT can be developed to be a low-cost, label-free and disposableDNA sensor since the effect of dsDNA is much bigger than that ofssDNA.

Acknowledgements

This work is financially supported by the Research Grant G-YF79and G-YH09 of the Hong Kong Polytechnic University.

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