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Ultrasensitive Detection of Dual Cancer Biomarkers with Integrated CMOS-Compatible Nanowire Arrays Na Lu, Anran Gao, Pengfei Dai, Hongju Mao, Xiaolei Zuo, Chunhai Fan, Yuelin Wang, and Tie Li Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b01729 • Publication Date (Web): 16 Oct 2015 Downloaded from http://pubs.acs.org on October 19, 2015
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Analytical Chemistry
Ultrasensitive Detection of Dual Cancer Biomarkers with Integrated CMOS-Compatible Nanowire Arrays Na Lu,† Anran Gao,† Pengfei Dai,† Hongju Mao, ‡ Xiaolei Zuo,§ Chunhai Fan,§ Yuelin Wang,† and Tie Li,*,† †
Science and Technology on Microsystem Laboratory, Shanghai Institute of Microsystem and Information Technology, Chinese Academy of Sciences, Shanghai 200050, China ‡ State Key Laboratory of Transducer Technology, Shanghai Institute of Microsystem and Information Technology, Chinese Academy of Sciences, Shanghai 200050, China §
Division of Physical Biology & Bioimaging Center, Shanghai Synchrotron Radiation Facility, CAS Key Laboratory of Interfacial Physics and Technology, Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai 201800, China ABSTRACT: A direct, rapid, highly sensitive, and specific biosensor for detection of cancer biomarkers is desirable in early diagnosis and prognosis of cancer. However, the existing methods of detecting cancer biomarkers suffer from poor sensitivity as well as the requirement of enzymatic labeling or nanoparticle conjugations. Here, we proposed a two-channel PDMS microfluidic integrated CMOS-compatible silicon nanowire (SiNW) field-effect transistor arrays with potentially single use for label-free and ultrasensitive electrical detection of cancer biomarkers. The integrated nanowire arrays showed not only ultrahigh sensitivity of cytokeratin 19 fragment (CYFRA21-1) and prostate specific antigen (PSA) with detection to at least 1 fg/mL in buffer solution, but also highly selectivity of discrimination from other similar cancer biomarkers. In addition, this method was used to detect both CYFRA21-1 and PSA real samples as low as 10 fg/mL in undiluted human serums. With its excellent properties and miniaturization, the integrated SiNW-FET device opens up great opportunities for point-of-care test (POCT) for quick screening and early diagnosis of cancer and other complex diseases.
Cancer is often silent in its early course and diagnosed at an advanced stage when treatment outcomes are not favorable.1 Among them, lung cancer continues to be the most common and lethal cancers, accounting for more than one in four of all cancer deaths in the US.2 Lung cancer is classified into two main types: nonsmall cell lung cancer (NSCLC) and small cell lung cancer (SCLC). NSCLC is much more common than SCLC. Prostate cancer is the most prevalent cancer and the second leading cause of death from cancer in men in America; moreover, the occurrence rate of prostate carcinoma increases greatly in older men (aged 65 or older) -more rapidly than any other cancer.3 Hence, quick screening in large area is known to be a crucial and effective way for early diagnosis of cancer to improve survival rates of patients. Biomarkers, as indicators of the state of disease, greatly improve early diagnosis and prognosis of cancer.4,5 CYFRA 21-1, soluble cytokeratin 19 (CK19) fragment, has proven to be the most sensitive in the diagnosis and prognostic of NSCLC,6 the level of which is associated with the disease process. Moreover, the detection of CYFRA21-1 may be helpful in identifying suspicious lung masses.7 Prostate specific antigen (PSA) is a glycoprotein, secreted by the epithelial cells of the prostate gland. Because of its absolute tissue specificity, PSA has been recognized as the most commonly used tumor marker for screening of early prostate cancer and monitoring the recurrence after treatment.8,9 However, it is proven to be inadequate to make tests of most single biomarkers for diagnosis in many
cases. A panel of biomarkers can generally provide complementary information to enable effective disease treatment and improve survival rate.10,11 Although a variety of strategies have been developed for detection of cancer biomarkers, including enzyme-linked immunosorbent assay (ELISA),12,13 chemiluminescence methods,14 electrochemical biosensors,15,16 surface plasmon resonance (SPR),17 and nanomaterials-based methods,18,19 they are often limited by sophisticated instrumentation, poor sensitivity, and labor-intensive labeling steps. Therefore, it is highly urgent and utmost significant to develop simple, rapid, high sensitive and selective detection techniques for determination of biomarkers in early cancer diagnosis. Owing to its exceptional electrical properties and excellent biocompatibility, silicon nanowire (SiNW) has been successfully applied to the detection of biological species via fieldeffect transistor (FET). For example, SiNWs have been employed for the detection of DNA,20-22 microRNAs,23,24 proteins,25-32 viruse particle,33 and cells.34,35 Currently, SiNWs is usually fabricated by either bottom-up and top-down techniques. Top-down fabricated SiNWs21,22,26,29,32,36 are compatible with standard fabrication technologies, which have advantages in the development of massive production and integrated system of point-of-care (POC) devices. Meanwhile, advances in microfluidics have employed a microchannel to delivery sample,41 enabling efficient transport of minute amount of sample onto the antibody-functionalized
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Analytical Chemistry
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nanowire surface. Integration of microfluidic channels into the sensor architecture has represented a critical innovation toward the realization of truly portable POC devices.42 Zheng et al. reported nanowire sensor arrays with microfluidic channels for highly sensitive and selective multiplexed detection of cancer markers to at least 0.9 pg/mL in serum samples.43 Stern et al. detected PSA and carbohydrate antigen 15.3 (CA15.3) from whole blood in physiological solutions by microfluidic integrated silicon nanoribbon based biosensors.44 However, in the NW-based biosensors, some of the microchannels are fabricated of polymers26,28,30,45 suffered from difficulty of microfabrication, and external pumping devices. On the other hand, most reported SiNW arrays were fabricated by bottom-up approach,25,46 deep ultra-violet (DUV) lithography,28,36 or electron beam (e-beam) lithography,29,39,40 which remains challenging in terms of commercialization or manufacturing cost. To address these challenges, we developed a two-channel PDMS microfluidic integrated CMOS-compatible SiNW arrays allowing for label-free and rapid electrical detection of dual cancer biomarkers with high selectivity and sensitivity. SiNWs were fabricated by a top-down approach via anisotropic wet etching and conventional photolithography with potential for massive production. The nanosensor showed high sensitivity for CYFRA21-1 and PSA with detection to as low as 1 fg/mL in buffer solution, as well as good selectivity for other cancer biomarkers. In addition, we carried out direct measurements of cancer markers in undiluted serum samples at femtogram concentrations. The simple, rapid, ultrasensitive, and single use SiNW-FET biosensor has a promising application for early diagnosis in cancer and other complex diseases. EXPERIMENTAL SECTION Materials. 3-Aminopropyltriethoxysilane (APTES) was purchased from Acros Organics (Geel, Belgium). Glutaraldehyde was purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Bovine serum albumin (BSA) was purchased from Sigma-Aldrich Co., LLC (CA, USA). CYFRA21-1 and CYFRA21-1 polyclonal antibody were purchased from Xema-Medica Co., Ltd. (Moscow, Russia) and Cal Bioreagents Inc. (CA, USA), respectively. PSA was obtained from US Biological Inc. (MA, USA). PSA monoclonal antibody, CEA and AFP were supplied by Fitzgerald Inc. (MA, USA). Ethanol and other chemicals were obtained from LingFeng Chemical Reagent Co., Ltd. (Shanghai, China). Phosphate buffered saline (1×PBS, pH 7.4) solution consists of 10 mM phosphate buffer, 0.14 M NaCl, and 2.7 mM KCl. Fabrication of SiNW Device. The SiNW array devices were produced by a CMOS-compatible top-down approach as previously reported.22 The SiNWs were fabricated on a 4 inch silicon-on-insulator (SOI) wafer. The wafers were p-doped with a light doping of 5×1015 cm-3. SiNWs were patterned by traditional photolithography and etched by anisotropic wet etching with tetramethylammonium hydroxide (TMAH). Then a high dose (1×1019 cm−3) of boron or phosphorus implantation was carried out to form an effective contact region. Ohmic contacts were realized by gold metallization. Finally, a sandwich structure of a nitride-oxide-nitride stack was deposited to protect the metal leading wires, leaving the nanowire sensors and metal pads exposed. Bonding of the PDMS Microchannels and the SiNW Chips. A silicon molds with rectangular microchannels (500
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µm in width, and 100 µm in height) were fabricated by deep reactive ion etching (DRIE) for PDMS replication.47 Before bonding, PDMS samples were cleaned with ethanol and deionized water, and dried in nitrogen (N2) flow. Next, PDMS microchannels were modified using a 20% (w/v) polyvinylpyrrolidone (PVP) solution to improve the wetting properties of the PDMS channel and reduce non-specific sites. Then PDMS samples were activated by oxygen (O2) plasma under the optimal parameters (a flow rate of 500 mL/min, a exposure time of 30 s, and a RF of power 50 W) in a MVD 100 system (Applied MicroStructures Inc., USA). After the plasma treatment, bonding between PDMS layer and SiNW device were immediately performed. Surface Functionalization. A silane chemistry was used to terminate amino groups onto the surface of nanowire by a 2% APTES ethanol solution overnight. The SiNW chip was then washed with ethanol, and then blow-dried. Next, the chip was immersed in a solution of 2.5% glutaraldehyde for 2 h. After bonding with PDMS microfluidic layer, two antibodies against CYFRA21-1 and PSA (20 µg/mL, 1×PBS) were separately functionalized on the two microchannel surfaces for 2 h, respectively. The chip was washed with PBS buffer solution for three times. Finally, 1% bovine serum albumin (BSA) was injected into the microchannels to block active sites. Electrical Measurement. Real-time electrical measurements were carried out by a Keithley 4200 semiconductor parameter analyzer (Keithley Instruments Inc., Cleveland, Ohio). A drain-source voltage of 1 V and a gate voltage of −1 V were applied to the SiNW devices, and the current through the SiNW was measured. Cancer marker samples (CYFRA211 and PSA) with volumes of less than 2 µL were injected in a buffer of 0.01× PBS (pH 7.4) at room temperature. SiNW devices with widths of approximately 100 to 200 nm were used in the sensing experiments. Results and Discussion SiNW Device Fabrication. The SiNWs used in this work were produced by a top-down approach as previously reported protocols.22 SiNWs in array format were fabricated on 4 inch SOI wafers by using CMOS compatible technologies, including conventional optical lithography, anisotropic wet etching with TMAH and thermal oxidation procedures. The diameter of SiNW ranged from 20 to several hundreds of nanometers, which was controlled by etching time and self-limitation oxidation. A schematic illustration of the prototype FET device fabricated with SiNW array was shown in Figure 1a. The basic nanowire sensor chip consisted of two addressable SiNW arrays, which was suitable for sensing of dual analytes. In each array, well-ordered SiNWs shared the same common source and individual drain with 10 electrically addressable devices (Figure 1b). Figure 1c showed a scanning electron microscopic (SEM) micrograph of the SiNW devices with width of 80 nm and length of 25 µm.
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Analytical Chemistry
Figure 1. SiNW device fabrication. (a) Schemetc illustration of the prototype FET device fabricated with SiNW arrays. The source (S), drain (D), backgate (G), and buried oxide (BOX) are labeled. (b) Microscopic image of a fabricated SiNW array on the chip. Zoomed-in image of one addressable SiNW device element highlighted by the rectangular box. (c) SEM micrograph of a SiNW with 80 nm in width and 25 µm in length. It is very important of integration of microfluidic channels into the SiNW chip towards realization of truly lab-on-chip devices. A PDMS microchannel system not only enables sample with minute amount to be automatically delivered onto the sensor array by capillary action, but also prevents sample from liquid evaporation and mechanical shocks. Figure 2a illustrated the scheme of bonding procedure of two-channel PDMS microfluidic sample delivery system onto the SiNW-FET chip. Refer to our previous work,47 we employed the rectangular microchannels with optimized dimensions of 500 µm in width, 100 µm in height, and 3 mm in length. Pristine PDMS surface has hydrophobic nature and does not enable capillary action, which may cause nonspecific absorption and bubble formation. To overcome this limitation, a biocompatible polymer PVP solution was introduced into PDMS channels (Figure 2b), after that the contact angle of microchannel decreased and the channel surface became hydrophilic. In addition, O2 plasma activation was used to produce silanol terminations (Si-OH) on the surface of PDMS. Because of terminating with hydroxyl group (-OH), it made PDMS surface to be more hydrophilic and enabled leakage free after bonding withthe SiNW device (Figure 2b) . A photograph of a biosensor chip with integrated two-channel PDMS sample delivery system on the SiNW device was shown in Figure 2d. After bonding, the PDMS channel surfaces demonstrated capabilities of capillary action and enabled fluid exchange at a flow rate of ~1.7 mL/hour47 without need of external pumping devices, favorable in dual detection of POCT diagnosis.
Figure 2. Bonding of PDMS layer and SiNW-FET device. (a) Scheme of the bonding procedure of PDMS microchannel layer onto SiNW-FET device. PDMS microchannels were treated with PVP solution, and then PDMS layer was irradiated by plasma. (b) Optical image of a two-channel PDMS microfluidic layer. (c) Image of PDMS microfluidic system superimposed on SiNW arrays for fluid exchange. (d) Photograph of a whole PDMS integrated SiNW-FET chip. Detection of Dual Cancer Biomarkers. In order to test the capabilities of the integrated devices , we chose CYFRA21-1 and PSA as two typical cancer biomarkers, which have been recognized as characteristics and predicators in early screening and clinical diagnosis of lung cancer and prostate cancer, respectively. Rapid, label-free, and ultrasensitive electrical detection of CYFRA 21-1 and PSA was carried out by using the microfluidic integrated SiNW chips. The sensing processes were illustrated in Figure 3. Firstly, a silane chemistry, as described earlier, was used to generate amino terminals on the nanowire surface, then the amine group is converted to aldehyde group by glutaraldehyde. Following, specific antibodies of CYFRA21-1 and PSA were efficiently cross-linked on the surface of different channels via amide condensation reaction, respectively. Finally, BSA solution was introduced on the surface to reduce non-specific absorption. We used p-type silicon nanowire arrays integrated with PDMS layer for dual detection of CYFRA21-1 and PSA. Current-versustime data was recorded after introduction of cancer markers. Measurements showed that the current increased stepwise upon delivery 10 pg/mL of CYFRA21-1, and that the current value was maintained after the introduction of pure buffer solution (Figure 3b). A rapid increase in current occured upon addition of CYFRA21-1, which was in good agreement that CYFRA21-1 (pI 4.6-4.8)48 was negatively charged at the pH of our measurements, leading to binding of a negatively charged species and the depletion of carriers of the p-type nanowire. Similarly, introduction of 1 pg/mL of PSA also produced a large current change based on anti-PSA immobilized, p-type nanowire devices, as shown in Figure 3c. The change in current was consistent with binding of a negatively charged species, as expected from the pI of PSA (6.8),9 and the pH of buffer solution (pH7.4). These results demonstrated rapid (
