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Final pg. 1 Final for! The exam is worth 60 points – roughly two problem sets. Ground Rules: 1. Unlike the problem sets, no collaboration with fellow students (or anyone else). 2. Unlike the problem sets, you should NOT use the internet to answer the questions (with the exception of one link provided in question #4). You may use any course material or any chemistry/biology textbook that is useful. 3. Please do not directly copy sentences from the attached paper or from the textbook in your answers. This is plagiarism. Write your answers in your own words. 4. Please feel free to reach out to me directly (hartley@ku.edu) if anything is ambiguous or you have questions. By signing/typing your name below, you are agreeing that you will follow the above ground rules. 1. (2 points) Final pg. 2 2. (9 points) Choose a video from your classmates’ videos on Blackboard that you have NOT already watched for the discussion (e.g., if you are in Group 1, choose a video from Group 5-8). Also, please choose a video on a topic different than your group’s topic. Watch the video and answer the following questions. For reference: Group 1: Atomic Absorption Spectroscopy Group 2: NMR Group 3: MRI Group 4: GC Group 5: GC-MS Group 6: Atomic Absorption Spectroscopy Group 7: NMR Group 8: GC a. Which group did you watch? (1 point) b. List three things you learned from the video. (3 points) c. In the research application discussed in the video, what question is being addressed by the featured instrumentation? (2 points) d. Identify another method that could be used to address the same question. List at least two advantages or disadvantages the alternative method has over the method in the video. (3 points) Final pg. 3 3. (10 points) Laser scanning confocal microscopy (LSCM) and scanning electron microscopy (SEM) are both surface methods that work by “raster” scanning, in which the sample is scanned line by line and data is collected at each point to generate an image. a. For each instrument, what is the source (primary beam) and what is detected (secondary beam)? (2 points) b. For each instrument, state whether the source is in direct line with the sample or at a 90° angle. Provide a short rationale for this instrumentation setup. (4 points) If your sample is a brain slice and you put it directly into either the LSM or SEM, very little signal would be detected. c. Explain why there is no signal for both methods. (2 points) d. Explain how you would prepare the sample to generate an observable signal for both methods. (2 points) Final pg. 4 4. (22 points) Plastic waste in the oceans is a major environmental problem. Contaminants that originate from plastics and are found in marine waters include phthalates. Phthalates represent a family of compounds with different modifications (R’s) on the ester groups. A common phthalate, diethylphthalate, is shown. In this experiment, your goal is to develop a quantitative assay to quantify phthalates in ocean water samples. First you will develop a method based on mass spectrometry. a. What type of ionization method and mass analyzer will you use for this method? (2 points) b. Why did you make the choices that you did in a? (1 point) c. Do you need to couple it to a separation method (GC or LC) to detect phthalates in ocean samples? (1 point) d. If c is yes, which one and why? If c is no, explain your answer. (1 point) Phthalates are typically ~1000 – 15,000 ng/L in ocean water. e. Choose a calibration method for your experiment (external curve, standard addition, or internal addition). (1 point) f. State which standard(s) you need for your chosen calibration method – be specific. (1 point) g. Your calibration curve should have at least 6 points. Please state what concentrations you will choose for your calibration curve points and if applicable, what concentration you will choose for an internal standard. (2 points) A mass spectrometry method involving fragmentation would be useful to identify phthalates that were previous not known to be in ocean water. h. Name two methods of mass spectrometry that involve compound fragmentation. (2 points) i. What is the difference between a product-ion spectra and a precursor-ion spectra? (2 points) j. Assume the phthalates fragment such that the R-groups from the esters are cleaved. Would a product-ion or precursor-ion scan be more useful for identifying previously unknown phthalates? (1 point) k. Explain your answer in j. (2 points) For the following alternative methods, state one advantage or one disadvantage (you choose which!) that the method would have compared to the method you developed above. Consider that you will be measuring the phthalates in a complex ocean water sample. Also, do not use the same answer twice (even if it applies to both)! l. Electrochemical method for detecting diethylphthalate (2 points) m. Capillary electrophoresis coupled to MS detection (2 points) n. Molecular sensor that emits fluorescence upon binding to diethylphthalate (2 points) Final pg. 5 5. (17 points) The attached paper uses the biological technique of PCR (polymerase chain reaction) with regards to a SARS-coronavirus – but not COVID-19 - as you will see this paper predates our current crisis. Although you do not need to understand the details of PCR to answer these questions, if you would like to know more about PCR while completing this exam, you can visit the tutorial at this website: https://www.khanacademy.org/science/ap-biology/gene-expression-andregulation/biotechnology/a/polymerase-chain-reaction-pcr A friendly reminder – please do not directly quote from the paper in your answer. This constitutes plagiarism. Please write your answers in your own words. a. What is the gap in technology being addressed by this paper? (2 points) b. What instrumental method(s) are being used to address the challenge? (2 points) c. What material and general fabrication methods were used to prepare the device? (2 points) d. How was the sample loaded onto the device? (1 point) e. The instrument diagram in Figure 3 has two major parts - one for reaction/separation and one for detection. Identify all modular components of both parts (i.e., sources, wavelength selectors, sample holders, transducers, and/or signal readout modules). (4 points) f. Which figures of merit are mentioned in the paper and what are the values? (3 points) g. Do the authors demonstrate that they have developed an improved technology? (1 point) h. Provide support for your answer in part g using data from the paper. (2 points) 3032 Xiaomian Zhou1 Dayu Liu1 Runtao Zhong1 Zhongpeng Dai1 Dapeng Wu1 Hui Wang1 Yuguang Du1 Zhinan Xia2 Liping Zhang3 Xiaodai Mei1 Bingcheng Lin1 1 Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, P. R. China 2 Dana Fabre Cancer Research Institute, Harvard Medical School, Longwood, Boston, MA, USA 3 Genomic Department, Wyeth Pharmaceuticals, Inc., Cambridge, MA, USA Electrophoresis 2004, 25, 3032–3039 Determination of SARS-coronavirus by a microfluidic chip system We have developed a new experimental system based on a microfluidic chip to determine severe acute respiratory syndrome coronavirus (SARS-CoV). The system includes a laser-induced fluorescence microfluidic chip analyzer, a glass microchip for both polymerase chain reaction (PCR) and capillary electrophoresis, a chip thermal cycler based on dual Peltier thermoelectric elements, a reverse transcription-polymerase chain reaction (RT-PCR) SARS diagnostic kit, and a DNA electrophoretic sizing kit. The system allows efficient cDNA amplification of SARS-CoV followed by electrophoretic sizing and detection on the same chip. To enhance the reliability of RT-PCR on SARS-CoV detection, duplex PCR was developed on the microchip. The assay was carried out on a home-made microfluidic chip system. The positive and the negative control were cDNA fragments of SARS-CoV and parainfluenza virus, respectively. The test results showed that 17 positive samples were obtained among 18 samples of nasopharyngeal swabs from clinically diagnosed SARS patients. However, 12 positive results from the same 18 samples were obtained by the conventional RT-PCR with agarose gel electrophoresis detection. The SARS virus species can be analyzed with high positive rate and rapidity on the microfluidic chip system. Keywords: Microfluidic chip system / Miniaturization / Multiplex reverse transcription-polymerase chain reaction / On-line chip polymerase chain reaction / Severe acute respiratory syndrome DOI 10.1002/elps.200305966 1 Introduction The severe acute respiratory syndrome (SARS), which is an acute respiratory illness caused by a new coronavirus, started to spread around the world in the early spring of 2003 [1]. It is the first major new infectious disease of this century, unusual in its high morbidity and mortality rates. Until now, more than 8000 persons with probable SARS have been diagnosed, 916 patients died. Fortunately, the outbreaks in the initial waves of infection have been brought under control. In the absence of effective drugs or a vaccine for SARS, control of this disease relies on the rapid identification of cases and their appropriate management, including the isolation of suspect and probable cases and the management of their close contacts. Correspondence: Dr. Bingcheng Lin, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, P. R. China E-mail: bclin@dicp.ac.cn Fax: 186-0411-84379065 Abbreviations: HPMC, hydroxyprolymethylcellulose; RT-PCR, reverse transcription-polymerase chain reaction; SARS-CoV, severe acute respiratory syndrome-associated coronavirus; TBE, Tris-borate-EDTA buffer ? 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Therefore, the establishment of a rapid noninvasive test for this virus is a high priority for detection and control of this disease. Due to the efforts of the WHO-led international multicenter collaborative network of laboratory testing for SARS, tests for the novel coronavirus have been developed with unprecedented speed [2]. The methods include a molecular test reverse transcription ((RT)-PCR) [3], virus isolation, antibody detection including enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay (IFA), and a neutralization test. But, as SARS epidemic spreads, the specific, rapid, and practical diagnostic tests will become increasingly critical, both for the control of the epidemic and for the management of patients. Among tests, antibody tests detect antibodies produced in response to the SARS coronavirus infection. The determination of different types of antibodies (immunoglobulin M (IgM) and IgG) after SARS-coronavirus (CoV) infection offers a good diagnostic method. However, antibodies apparently increase about 10 days after infection, so the pathogen is undetectable at the early stage of infection. The other effective method is virus isolation in specimens (such as respiratory secretions, blood or stool) from SARS patients, which can be detected by inoculating cell cul- SARS-coronavirus detection on a microfluidic chip system tures and growing the virus. After isolation, the virus still needs to be identified as SARS virus with further tests. Positive cell culture results indicate the presence of live SARS-CoV in the sample tested and negative cell culture results do not exclude SARS. Cell culture methods are time-consuming and nonspecific. It is unlikely accepted as a universal SARS detection method. The molecular test (RT-PCR) is one of the most commonly used methods for RNA virus detection; it has high sensitivity and can make diagnosis at the early stage [4]. Poon et al. [5] reported a real-time quantitative PCR assay, which performed quite well (sensitivity of 79% and specificity of 98%) and it appeared to become positive before antibodies first appeared. It was a good early diagnostic assay for SARS but it was expensive for people in the developing countries. The conventional RT-PCR assay is cheap but the positive rate is lower than the quantitative PCR assays [6]. Therefore, an ideal test for virus will not be only rapid, sensitive, and specific, but also inexpensive and technologically simple, so that it is available at the point of care even in small hospitals or in communities in the developing countries. To achieve the goal, therefore, it is worth studying whether or not the microfluidic chip (lab-on-a-chip) technology can play a role in the early identification of contagion, especially infection of SARSCoV. The miniaturization of chemical and mechanical devices for microelectromechanical systems has gained great attention in industry and academic institutions worldwide over recent years. Examples include lab-on-a-chip or microfluidic devices such as on-chip flow-through PCR, microreaction technology, electrophoretic separation devices, etc. [7]. These approaches offer novel ways to achieve fast separation with high resolution in a miniaturized fabrication including sample pretreatment steps i.e., sample concentration, labeling, and digestion with the additional possibility of multiplexing. At the same time, there are a lot of alternative methods of DNA analysis on microchips. For example, the TaqMan real-time PCR in silicon-based reactors amplified a variety of DNA and RNA targets [8–10] in order to detect pathogenic viruses and bacteria, plant genes, human genetic diseases, and single nucleotide polymorphism [11, 12]. Although these alternative methods do not provide the DNA sizing information required for a number of biological, biomedical, and forensic analyses, they are useful for various high-throughput applications. The high sensitivity of DNA analysis (detection limit in the order of 10221 mol) [13] had been achieved among those various approaches. Particularly, on-chip PCR provided both rapid, efficient amplification of DNA and low cost [14], and even followed by electrophoretic analysis of the products in situ [15]. So, we assume that microfluidic chip ? 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 3033 technology may play an important role in the determination of the SARS-CoV of specimens. In this paper, we report a microfluidic chip system to detect this virus in clinical specimens. 2 Materials and methods 2.1 Chemicals A 100 mmol/L Tris (Sigma Chemical, St. Louis, MO, USA)/ 100 mmol/L boric acid/2 mmol/L EDTA buffer (TBE)/2.0% hydroxyprolymethylcellulose (HPMC, 50 cps; Sigma) with a pH of 8.5 was used for separations of 50 ng/mL F X-174/HaeIII digest DNA restriction fragments ranging in size from 72 to 1353 bp (TaKaRa Biotechnology, Dalian, China). An approximate 130 ng/mL 100 bp DNA ladder marker ranging in size from 100 to 1500 bp, which is diluted 20-fold in the run buffer, was also purchased from TaKaRa Biotechnology, as well as the PCR products of SARS-CoV. SYTOX Orange nucleic acid stain was obtained from Molecular Probes (Eugene, OR, USA). A 1 mmol/L dye solution was prepared in 2.0% HPMC-TBE buffer in order to label DNA on-line. Polyvinylpyrrolidone (PVP) was from Acros (Pittsburgh, PA, USA). A 4.02 mM stock solution of Rhodamine 6G dye (Sigma) was prepared in a 100 mM Tris/100 mM boric acid/2 mM EDTA (TBE) buffer, pH 9. Further solutions were prepared by serial dilution of the 4.02 mM fluorescein stock solution with the same running buffer for determining the detection limit. All buffers were prepared in doubly distilled water. Solutions were filtered (0.22 mm filters) before introduction into the chip. 2.2 Samples The in vitro cultured SARS sample was obtained from the autopsied lung tissue of a deceased patient, in whom SARS was diagnosed according to the WHO guidelines [2]. The sample was cultured with Vero-E6 cell line. Total SARS-CoV RNA was isolated from a 210 mL cell culture. Eighteen samples of nasopharyngeal swabs of clinically diagnosed SARS patients were from the Nanfang Hospital of the First Military Medical University in Guangzhou. Samples were kept under 2207C until analysis. The positive control of SARS-CoV is the cDNA fragment of the SARS-CoV sequence, which was provided by BNI (Hamburg, Germany). The negative control of SARS-CoV is the parainfluenza virus. RNA of SARS-CoV was extracted with RNAeasy Mini Kit (Qiagen, Hilden, Germany) before performing the RTPCR reaction. Miniaturization Electrophoresis 2004, 25, 3032–3039 3034 X. Zhou et al. Electrophoresis 2004, 25, 3032–3039 2.3 RT-PCR 2.3.1 RT of RNA Total RNA from Vero-E6 cell cultures and nasopharyngeal swab samples of clinically diagnosed SARS patients were reversely transcribed to cDNA. Reverse transcription was performed on a GeneAmp PCR system 2400 (Perkin-Elmer, Norwalk, CT, USA) and all reagents used for RT were from TaKaRa Biotech. Two mL total RNA from cell cultured SARS sample was added to the following RT reaction mixture (20 mL) containing: 4 mL Mg21 (25 mmol/L), 2 mL 106RNA PCR buffer containing 2.5% PVP, 7.5 mL RNase-free distilled H2O, 2 mL dNTP mixture (10 mmol/L each), 0.5 mL RNase inhibitor (40 U/mL), 1 mL AMV reverse transcriptase XL (5 U/mL), and 1 mL Oligo dT-adapter Primer (2.5 pmol/mL). The RT reaction was performed under the following conditions: 10 min at 307C, 30 min at 507C, 5 min at 997C, and 5 min at 47C. 2.3.2 Conventional PCR The two primer sets of duplex PCR, which were used to determine the cultured SARS-CoV and the samples of the clinically diagnosed SARS patients, were 5’-TAG GATTGCCTACGCAGACT-3’ and 5’-AGAGCCATGCCTAA CATGCT-3’ (for the 240 bp product), and 5’-ATTGGCT GTAACAGCTTGAC-3’ and 5’-TAGGGTAACCATTGACTT GG-3’ (for the 438 bp product), respectively. The duplex PCR mixture contained 2 mL of the reverse-transcribed product, 5 mL 106PCR buffer II, 0.5 mL LA Taq DNA polymerase, 1 mL (0.5 mL each of primers) duplex primers (20 mM), 5 mL Mg21 (25 mmol/L); sterile water was added to a total volume of 50 mL. The following PCR reaction protocol was employed: 947C for 5 min followed by 30 cycles at 947C for 30 s, 557C for 30 s, 727C for 30 s, and then 727C for 7 min. The products of PCR reaction were analyzed by visualizing with ethidium bromide staining. The reaction was finished on the GeneAmp PCR system 2400 (Perkin-Elmer). 2.4 Microchip fabrication The glass microchips were fabricated using standard photolithographic and wet chemical techniques as described elsewhere [16]. The microchannel design in Fig. 1 was transferred onto the substrates using a positive photoresist, photomask, and UV exposure. The microchannels were etched into the substrate in a dilute, stirred ? 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Figure 1. (a) Layout of the microfabricated channels; (b) PCR chip. The reactive area includes the reactive pool and the serpent-shape channel. The point of fluorescence detection is marked with an arrow. Potential of sample injection, 400 V/cm at the sample waste; grounding the sample reservoir for 30 s. The buffer and waste reservoirs had no potentials applied. Potentials of separation, 0, 1.1, 0.3, and 0.3 kV at the buffer, waste, sample, and sample waste reservoirs, respectively. HF/NH4F bath. To form the closed network of channels, a cover plate was bonded to the substrate over the etched channels by hydrolyzing the surfaces, bringing them into contact with each other, then processing thermally to 2007C in the vacuum oven for 1 h. Subsequently, the pair glass plates were placed between two pieces of smooth China plates and annealed in a Model SX3-4-10 programmable furnace (Zhonghuan Test Electrical Furance, Tianjin City, China). The temperature program was as follows: initial heating to 1007C for about 1 h, and to 6207C for about 3 h, followed by natural cooling of the furnace to room temperature. The channels were typically 20 mm deep and 50–60 mm wide at half-depth. The separation length was 3.0–3.5 cm. For forming reservoirs connected to the microchannels, the 2 mm diameter holes in the top glass plate (either substrate or cover plate) were drilled by the CK-250L ultrasound driller (Shantou Goldstar Ultrasonic Machinery, China). The typical volume of the reservoirs was 15 mL. Electrophoresis 2004, 25, 3032–3039 SARS-coronavirus detection on a microfluidic chip system 2.5 Microchip electrophoresis 3035 The electroosmotic flow in the channels was minimized using PVP-TBE buffer as a dynamic coating [17]. The channels were filled with a sieving buffer solution for microchip gel electrophoresis after they were oxidized in boiling H2SO4/H2O2 (1:1) for 10–15 min, followed by washing with deionized H2O. The coating was stable for multiple injection/separation cycles and provided reproducible separation performance. For DNA fragment sizing, 2.5% PVP solution in a 100 mM Tris/100 mM boric acid/2 mM EDTA buffer was used. An intercalating dye, SYTOX Orange nucleic acid stain, was added to the polymer solution at 1 mM for the PCR on-chip experiments. A DNA marker containing 11 DNA fragments of 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, and 1500 bp at approximately equal weight/volume concentration was used for PCR verification, product size determination, and estimations of the amplification efficiency. For the laser-induced fluorescence (LIF) detection, the LIF microfluidic chip analyzer was used. For the conventional injection/separation cycle, the sample was first loaded into the sample reservoir and then injected by applying 400 V/cm at the sample waste and grounding the sample reservoir for 30 s. The buffer and waste reservoirs had no potentials applied since the low diffusion coefficients of DNA fragments in the polymer sieving medium resulted in no substantial increase in the injection plug length or deterioration of separation channel. The relative potentials were switched to 0, 1.1, 0.3, and 0.3 kV at the buffer, waste, sample, and sample waste reservoirs, respectively. 1 inch, output power of 30 W). Aluminum cooling flakes are attached to the backside of the Peltier devices as heat sinks. A small cooling fan was also attached to each of the two aluminum blocks for convective cooling. During PCR thermal cycling, the 2/3 area of the entire chip was sandwiched between the two Peltier elements. The device temperature and Peltier surface temperature were monitored using thermocouples. Software inhouse made was used for thermal cycle temperature control. A drop of mineral oil on the bottom Peltier element ensured good thermal contact with the cycled region of the chip. All microchannels and reservoirs, excluding the PCR reaction area (including sample reservoir) in Fig. 1, were filled with sieving solution. The reaction well was first treated with 2.5 g/L bovine serum albumin (BSA) solution for 2–3 min to modify the inner glass surface of the milled reservoir. The BSA was then removed from the well and replaced with 3.5–4 mL of the PCR reaction mixture (individual or duplex PCR reaction mixture and cDNA of the sample). To prevent evaporation, all wells were topped with 6–7 mL of mineral oil. The temperature steps were 947C for 2 min, followed by 94, 55, and 727C with holding times of 45, 30, and 30 s for 30 cycles, and then 727C for 2 min to perform DNA amplification. The top thermoelectric element of the thermal cycler for the micro-PCR device was not placed in direct contact with the chip in order to eliminate the requirement of sealing the top reaction reservoir against contact-driven capillary wicking. Thus, the top assembly was positioned over the chip using 0.5 mm thick graphite strips as spacers. 2.6 Thermal cycling on-chip 2.7 Detection system Figure 2a shows a picture of the thermal cycler for the micro-PCR device, which was designed and built inhouse using two Peltier thermoelectric devices (1 inch6 Figure 3 shows schematically the layout of a micro-CE device with integrated confocal laser fluorescence detector and automicromanipulation stage. The dimensions Figure 2. (a) Thermal cycler for the micro-PCR device (home-made); (b) temperature cycling trace curve of the PCR chip. ? 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 3036 X. Zhou et al. Electrophoresis 2004, 25, 3032–3039 Figure 3. Schematic representation of the experimental instrument and the laser confocal detection arrangement. of the instrument were 50650645 cm. The output radiation (532 nm) from an air-cooled laser diode (LD)pumped solid-state laser (20 mW; Mektec Seiwa Corporation, Beijing, China) passes through a 532 nm filter (Omega Optical, Brattleboro, VT, USA). The beam is reflected by a dichroic beam-splitter (Omega Optical) and focused into the channel through a 206 microscope objective (0.4 N.A.). The emission signal is collected by the same objective and transmitted back through the dichroic beam-splitter. The emission beam passes through a bandpass filter (Omega Optical), which may be alternated easily to fit a wide selection of dyes, and is focused by a focusing lens through a 400 mm pinhole. The photomultiplier tube (R212; Japan) is mounted in an integrated detection module including high-voltage power supply, voltage divider, and amplifier. The chargecoupled device (CCD) camera was fixed at the same board as the photomultiplier tube in order to focus and observe the channel. The whole optical system was installed on the X-Y-Z translational stage (3-D micro- ? 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim manipulator, adjusting precision 1 mm), and the focus can be controlled via the picture displayed on the screen. 3 Results and discussion 3.1 Detection limit of the detection system The detection limit of Rhodamine 6G dye for the detection system with the home-made chip and a 100 mM TBE buffer is 6.67610213mol/L (S/N . 3). The linear range of detection was 4.0261026mol/L to 4.0261029mol/L (r = 0.9996). For detailed information we refer to the literature [18]. A 50 ng/mL F X-174/HaeIII digest DNA marker, with restriction fragments ranging in size from 72 to 1353 bp (72, 118, 194, 234, 271, 281, 310, 603, 872, 1078, 1353 bp), was serially diluted and determined on the detection system. When the S/N of the 603 bp fragment was 3, its corresponding concentration was Electrophoresis 2004, 25, 3032–3039 SARS-coronavirus detection on a microfluidic chip system 3037 0.2 ng/mL (the mass of the 603 bp fragment detected was 3.36 fg according to the injection volume). These data show that this detection system has a high sensitivity, which is over 100-fold higher than that of agarose gel electrophoresis in the conventional PCR assay [18]. The high assay sensitivity is important for early diagnosis of the diseases and early identifying of the suspected patients with SARS. 3.2 Design and optimization of SARS-CoV RT-PCR assay kit Primers were designed according to the conserved regions of open reading frame 1b (replicase 1B) following the Tor 2 SARS genome sequence in order to amplify sequences within two regions of SARS genome, 15240– 15612 and 17743–18349. Multiplex PCR is a modification of the basic PCR method, where multiple pairs of primers are used in the same reaction [19, 20]. A multiplex protocol appears to be useful for the investigation of different target sequences at the same time. By RT to cDNA and subsequent amplification, the RNA template can also be amplified by multiplex PCR. The SARS RNA genome is apt to mutation, which may lead to primers ill-matched with templates. Multiplex PCR, which can effectively reduce the possibility of false-negative PCR results caused by genome instability, amplifies different regions at the same time. So we designed nine pairs of primers according to the Tor 2 SARS genome sequence, and 2 of 9 pairs of primers were optimized as the primers for RT-PCR SARS-CoV assay kit. The sequences of two pairs of the primers are 5’-TAGGATTGCCTACGCAGACT-3’ and 5’-AGAGCCATGCCTAACATGCT-3’ (for the 240 bp product), and 5’-ATTGGCTGTAACAGCTTGAC-3’ and 5’-TAG GGTAACCATTGACTTGG-3’ (for the 438 bp product), respectively. The concentration of Mg21, enzymes, reaction time, cycling times, etc., for RT-PCR SARS-CoV assay kit were optimized. Optimization of RT-PCR SARS-CoV assay with SARS-CoV from the cultured Vero-E6 cells was finished by the GeneAmp PCR system 2400 (Perkin-Elmer) thermocycler. Reaction products from duplex PCR were analyzed by agarose gel electrophoresis (Fig. 4). 3.3 PCR-CE on-line A series of thermal transfer experiments have been conducted using different thicknesses of the glass (e.g., 2 mm, 0.5 mm, 0.15 mm), and it was found that the intrareservoir temperature would fit the set point temperature better by using a thinner glass cover (data not shown). However, the thinnest glass cover plate of the bottom of every reservoir is easily shattered, particularly during installation of the electrodes. Therefore, the thickness of the ? 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Figure 4. Agarose gel electropherogram of the products from duplex PCR. glass cover plate was kept thin (0.5 mm) in order to achieve a reasonable thermal cycle time. The temperature cycling of the PCR chip in a dual Peltier assembly is represented by trace curve in Fig. 2b, which shows the temperature profile for five cycles of the program inhouse software. The Peltier surface temperature and intra-reservoir temperature were measured by thermocouples. A built calibration chip contained an embedded thermocouple to assess the intra-reservoir temperature and compare it with the temperature of the Peltier surface. These data were used to design the thermal cycle profile of amplification experiments. The chips used in the experiments did not have the embedded thermocouple. Cycle times were initiated according to the intrareservoir temperature. Successful DNA amplification is dependent on the preliminary treatment of PCR reservoirs as well as on the reaction mixture volume. The pretreatment of PCR reservoirs was performed as described by Khandurina et al. [15]. Optimum thermal uniformity during heating/cooling cycles resulting in a good PCR efficiency was achieved using 3–4 mL PCR cocktail in 2 mm diameter reaction reservoirs and a serpent-shape channel. To achieve PCR amplification, a pair of PCR primers was first performed on the chip thermal cycler, and then the duplex PCR was manipulated. Figure 5a shows the electropherogram of 240 and 438 bp PCR products of the positive control of SARS-CoV. Amplification and electrophoretic analysis were on the same chip. The total analysis time was approximately 50–60 min including 30 thermal cycles and electrokinetic injection/separation manipulation. Figure 5b shows the electropherogram of the negative control (parainfluenza virus) and Fig. 5c the electropherogram of the 240 and 438 bp PCR products of the cultured SARSCoV. To identify the length of unknown amplified products or verify the sizes of known target sequences, PCR prod- 3038 X. Zhou et al. Electrophoresis 2004, 25, 3032–3039 Figure 5. Microfluidic chip electropherograms. (a) Positive control; (b) negative control; (c) cultured SARS-CoV (240 and 438 bp PCR products); (d) PCR product 1 100 bp DNA marker. ucts were mixed and coelectrophoresed with size markers of DNA as demonstrated in Fig. 5d for 240 and 438 bp products. The latter profile was obtained by simply adding a DNA marker solution to the sample reservoir containing the PCR product after mixing. Compared to the standard curve of the migration time vs. the fragment size of DNA marker, the size of the amplified DNA fragments was determined by their migration time. PCR with cDNA of the sample, the PCR chip was translated on the detection system to separate the PCR products. The chip-PCR and on-line CE separation for each sample were therefore performed on the chip (Fig. 1). Seventeen positives were achieved from 18 of the nasopharyngeal swabs from the clinically diagnosed patients with SARS by the microfluidic chip system. However, Fig. 6 shows that 12 positives of 18 SARS samples noted above were detected by the conventional PCR with agarose gel electrophoresis. 3.4 Determination of the clinical specimens All RNA of SARS-CoVs was extracted from 18 clinical SARS samples with RNAeasy Mini Kit (Qiagen) before performing the RT-PCR reaction. The RT reactions with the extracted RNA were performed on a conventional PCR cycler to get the cDNA of the sample. After chip- ? 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 4 Concluding remarks A microfluidic chip system was developed and successfully used in determining the SARS-CoV of specimens from clinically diagnosed SARS patients. The positive Electrophoresis 2004, 25, 3032–3039 SARS-coronavirus detection on a microfluidic chip system 3039 5 References Figure 6. Gel electropherogram of the positive products of RT-duplex PCR for the nasopharyngeal swabs of 18 clinically diagnosed patients with SARS. rate of the SARS-CoV for clinical SARS samples is up to 94.44% (17/18) using this system. The positive rate achieved by the conventional RT-PCR with agarose gel electrophoresis system was only 66.67% (12/18). Compared to the conventional system, the microfluidic chip system showed a higher positive rate (.27%), shorter testing time, and the potential application of determining the SARS-CoV of specimens. The authors thank the First Military Medical University for providing samples of clinically diagnosed SARS patients. This research was sponsored by the National Science Council of the People’s Republic of China under contract Nos. 20275039, 20035010, and 20299030, and in part by the Chinese Academy of Sciences for building the microfluidic chip analyzer and by the Dalian Institute of Chemical Physics, Chinese Academy of Sciences for studying SARS. Received December 30, 2003 ? 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim [1] WHO Summary table of SARS cases by country, 1 November 2002–7 August 2003, www.who.int/csr/sars/country/ 2003_08_15/en/. [2] SARS: Laboratory diagnosis tests. 29 April 2003, http:// www.who.int/csr/sars/diagnostictests/en. [3] McIntosh, K., Clin. Chem. 2003, 49, 845–846. [4] Hoard, J. C., Schultz, T. F., in: Collins, M., Clifton, N. J. (Eds.), Methods in Molecular Biology, Humana Press, Totowa, NJ 1991, Vol. 8. [5] Poon, L. L. M., Wong, O. K., Luk, W., Yuen, K. Y., Peiris, J. S. M., Guan, Y., Clin. Chem. 2003, 49, 845–846. [6] Wu, X. W., Cheng, G., Di, B., Yin, A. H., He, Y. S., Wang, M., Zhou, X.Y., He, L. J., Luo, K., Du, L., Chin. Med. J. 2003, 116, 988–990. [7] Freemantal, M., Chem. Eng. News 1999, 22, 27–36. [8] Northrup, M. A., Benett, B., Hadley, D., Long, G., Landre, P., Lehew, S., Richards, J., Stratton, P., Anal. Chem. 1998, 70, 918–992. [9] Belgrader, P., Benett, W., Hadley, D., Long, G., Mariella, R., Milanovich, F., Nelson, W., Richards, J., Stratton, P., Clin. Chem. 1998, 44, 2191–2194. [10] Belgrader, P., Benett, W., Hadley, D., Richards, J., Stratton, P., Mariella, R., Milanovich, F., Science 1999, 284, 449–450. [11] Ibrahim, M., Lofts, R., Jahrling, P., Henchal, E., Weedn, V., Northrup, M. A., Belgrader, P., Anal. Chem. 1998, 70, 2013– 2017. [12] Belgrader, P., Smith, J., Weedn, V., Northrup, M. A. J., Forensic Sci. 1998, 43, 315–319. [13] Effenhauser, C. S., Bruin, G. J. M., Paulus, A., Ehrat, M., Anal. Chem. 1997, 69, 3451–3457. [14] Yang, J., Liu, Y. J., Rauch, C. B., Stevens, R. H., Liu, R. L., Lenigk, R., Grodzinski, P., Lab Chip. 2002, 2, 179–187. [15] Khandurina, J., Mcknight, T. E., Jacobson, S. C., Waters, L. C., Foote, R. S., Ramsey, J. M., Anal. Chem. 2000, 72, 2995–3000. [16] Woolley, A. T., Mathies, R. A., Proc. Natl. Acad. Sci. USA 1994, 91, 11348. [17] Kopp, M. U., deMello, A. J., Manz, A., Science 1998, 280, 1046–1047. [18] Zhou, X. M., Dai, Z. P., Luo, Y., Liu, X., Wang, H., Lin, B. C., Chem. J. Chin. Univ. 2004, 3, 336–340. [19] Valassina, M., Cuppone, A. M., Cusi, M. G., Valensin, P. E., Clin. Diagn. Virol. 1997, 8, 227–232. [20] Cassinotti, P., Mietz, H., Siegl, G., J. Med. Virol. 1996, 50, 75–81. 3032 Xiaomian Zhou1 Dayu Liu1 Runtao Zhong1 Zhongpeng Dai1 Dapeng Wu1 Hui Wang1 Yuguang Du1 Zhinan Xia2 Liping Zhang3 Xiaodai Mei1 Bingcheng Lin1 1 Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, P. R. China 2 Dana Fabre Cancer Research Institute, Harvard Medical School, Longwood, Boston, MA, USA 3 Genomic Department, Wyeth Pharmaceuticals, Inc., Cambridge, MA, USA Electrophoresis 2004, 25, 3032–3039 Determination of SARS-coronavirus by a microfluidic chip system We have developed a new experimental system based on a microfluidic chip to determine severe acute respiratory syndrome coronavirus (SARS-CoV). The system includes a laser-induced fluorescence microfluidic chip analyzer, a glass microchip for both polymerase chain reaction (PCR) and capillary electrophoresis, a chip thermal cycler based on dual Peltier thermoelectric elements, a reverse transcription-polymerase chain reaction (RT-PCR) SARS diagnostic kit, and a DNA electrophoretic sizing kit. The system allows efficient cDNA amplification of SARS-CoV followed by electrophoretic sizing and detection on the same chip. To enhance the reliability of RT-PCR on SARS-CoV detection, duplex PCR was developed on the microchip. The assay was carried out on a home-made microfluidic chip system. The positive and the negative control were cDNA fragments of SARS-CoV and parainfluenza virus, respectively. The test results showed that 17 positive samples were obtained among 18 samples of nasopharyngeal swabs from clinically diagnosed SARS patients. However, 12 positive results from the same 18 samples were obtained by the conventional RT-PCR with agarose gel electrophoresis detection. The SARS virus species can be analyzed with high positive rate and rapidity on the microfluidic chip system. Keywords: Microfluidic chip system / Miniaturization / Multiplex reverse transcription-polymerase chain reaction / On-line chip polymerase chain reaction / Severe acute respiratory syndrome DOI 10.1002/elps.200305966 1 Introduction The severe acute respiratory syndrome (SARS), which is an acute respiratory illness caused by a new coronavirus, started to spread around the world in the early spring of 2003 [1]. It is the first major new infectious disease of this century, unusual in its high morbidity and mortality rates. Until now, more than 8000 persons with probable SARS have been diagnosed, 916 patients died. Fortunately, the outbreaks in the initial waves of infection have been brought under control. In the absence of effective drugs or a vaccine for SARS, control of this disease relies on the rapid identification of cases and their appropriate management, including the isolation of suspect and probable cases and the management of their close contacts. Correspondence: Dr. Bingcheng Lin, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, P. R. China E-mail: bclin@dicp.ac.cn Fax: 186-0411-84379065 Abbreviations: HPMC, hydroxyprolymethylcellulose; RT-PCR, reverse transcription-polymerase chain reaction; SARS-CoV, severe acute respiratory syndrome-associated coronavirus; TBE, Tris-borate-EDTA buffer ? 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Therefore, the establishment of a rapid noninvasive test for this virus is a high priority for detection and control of this disease. Due to the efforts of the WHO-led international multicenter collaborative network of laboratory testing for SARS, tests for the novel coronavirus have been developed with unprecedented speed [2]. The methods include a molecular test reverse transcription ((RT)-PCR) [3], virus isolation, antibody detection including enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay (IFA), and a neutralization test. But, as SARS epidemic spreads, the specific, rapid, and practical diagnostic tests will become increasingly critical, both for the control of the epidemic and for the management of patients. Among tests, antibody tests detect antibodies produced in response to the SARS coronavirus infection. The determination of different types of antibodies (immunoglobulin M (IgM) and IgG) after SARS-coronavirus (CoV) infection offers a good diagnostic method. However, antibodies apparently increase about 10 days after infection, so the pathogen is undetectable at the early stage of infection. The other effective method is virus isolation in specimens (such as respiratory secretions, blood or stool) from SARS patients, which can be detected by inoculating cell cul- SARS-coronavirus detection on a microfluidic chip system tures and growing the virus. After isolation, the virus still needs to be identified as SARS virus with further tests. Positive cell culture results indicate the presence of live SARS-CoV in the sample tested and negative cell culture results do not exclude SARS. Cell culture methods are time-consuming and nonspecific. It is unlikely accepted as a universal SARS detection method. The molecular test (RT-PCR) is one of the most commonly used methods for RNA virus detection; it has high sensitivity and can make diagnosis at the early stage [4]. Poon et al. [5] reported a real-time quantitative PCR assay, which performed quite well (sensitivity of 79% and specificity of 98%) and it appeared to become positive before antibodies first appeared. It was a good early diagnostic assay for SARS but it was expensive for people in the developing countries. The conventional RT-PCR assay is cheap but the positive rate is lower than the quantitative PCR assays [6]. Therefore, an ideal test for virus will not be only rapid, sensitive, and specific, but also inexpensive and technologically simple, so that it is available at the point of care even in small hospitals or in communities in the developing countries. To achieve the goal, therefore, it is worth studying whether or not the microfluidic chip (lab-on-a-chip) technology can play a role in the early identification of contagion, especially infection of SARSCoV. The miniaturization of chemical and mechanical devices for microelectromechanical systems has gained great attention in industry and academic institutions worldwide over recent years. Examples include lab-on-a-chip or microfluidic devices such as on-chip flow-through PCR, microreaction technology, electrophoretic separation devices, etc. [7]. These approaches offer novel ways to achieve fast separation with high resolution in a miniaturized fabrication including sample pretreatment steps i.e., sample concentration, labeling, and digestion with the additional possibility of multiplexing. At the same time, there are a lot of alternative methods of DNA analysis on microchips. For example, the TaqMan real-time PCR in silicon-based reactors amplified a variety of DNA and RNA targets [8–10] in order to detect pathogenic viruses and bacteria, plant genes, human genetic diseases, and single nucleotide polymorphism [11, 12]. Although these alternative methods do not provide the DNA sizing information required for a number of biological, biomedical, and forensic analyses, they are useful for various high-throughput applications. The high sensitivity of DNA analysis (detection limit in the order of 10221 mol) [13] had been achieved among those various approaches. Particularly, on-chip PCR provided both rapid, efficient amplification of DNA and low cost [14], and even followed by electrophoretic analysis of the products in situ [15]. So, we assume that microfluidic chip ? 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 3033 technology may play an important role in the determination of the SARS-CoV of specimens. In this paper, we report a microfluidic chip system to detect this virus in clinical specimens. 2 Materials and methods 2.1 Chemicals A 100 mmol/L Tris (Sigma Chemical, St. Louis, MO, USA)/ 100 mmol/L boric acid/2 mmol/L EDTA buffer (TBE)/2.0% hydroxyprolymethylcellulose (HPMC, 50 cps; Sigma) with a pH of 8.5 was used for separations of 50 ng/mL F X-174/HaeIII digest DNA restriction fragments ranging in size from 72 to 1353 bp (TaKaRa Biotechnology, Dalian, China). An approximate 130 ng/mL 100 bp DNA ladder marker ranging in size from 100 to 1500 bp, which is diluted 20-fold in the run buffer, was also purchased from TaKaRa Biotechnology, as well as the PCR products of SARS-CoV. SYTOX Orange nucleic acid stain was obtained from Molecular Probes (Eugene, OR, USA). A 1 mmol/L dye solution was prepared in 2.0% HPMC-TBE buffer in order to label DNA on-line. Polyvinylpyrrolidone (PVP) was from Acros (Pittsburgh, PA, USA). A 4.02 mM stock solution of Rhodamine 6G dye (Sigma) was prepared in a 100 mM Tris/100 mM boric acid/2 mM EDTA (TBE) buffer, pH 9. Further solutions were prepared by serial dilution of the 4.02 mM fluorescein stock solution with the same running buffer for determining the detection limit. All buffers were prepared in doubly distilled water. Solutions were filtered (0.22 mm filters) before introduction into the chip. 2.2 Samples The in vitro cultured SARS sample was obtained from the autopsied lung tissue of a deceased patient, in whom SARS was diagnosed according to the WHO guidelines [2]. The sample was cultured with Vero-E6 cell line. Total SARS-CoV RNA was isolated from a 210 mL cell culture. Eighteen samples of nasopharyngeal swabs of clinically diagnosed SARS patients were from the Nanfang Hospital of the First Military Medical University in Guangzhou. Samples were kept under 2207C until analysis. The positive control of SARS-CoV is the cDNA fragment of the SARS-CoV sequence, which was provided by BNI (Hamburg, Germany). The negative control of SARS-CoV is the parainfluenza virus. RNA of SARS-CoV was extracted with RNAeasy Mini Kit (Qiagen, Hilden, Germany) before performing the RTPCR reaction. Miniaturization Electrophoresis 2004, 25, 3032–3039 3034 X. Zhou et al. Electrophoresis 2004, 25, 3032–3039 2.3 RT-PCR 2.3.1 RT of RNA Total RNA from Vero-E6 cell cultures and nasopharyngeal swab samples of clinically diagnosed SARS patients were reversely transcribed to cDNA. Reverse transcription was performed on a GeneAmp PCR system 2400 (Perkin-Elmer, Norwalk, CT, USA) and all reagents used for RT were from TaKaRa Biotech. Two mL total RNA from cell cultured SARS sample was added to the following RT reaction mixture (20 mL) containing: 4 mL Mg21 (25 mmol/L), 2 mL 106RNA PCR buffer containing 2.5% PVP, 7.5 mL RNase-free distilled H2O, 2 mL dNTP mixture (10 mmol/L each), 0.5 mL RNase inhibitor (40 U/mL), 1 mL AMV reverse transcriptase XL (5 U/mL), and 1 mL Oligo dT-adapter Primer (2.5 pmol/mL). The RT reaction was performed under the following conditions: 10 min at 307C, 30 min at 507C, 5 min at 997C, and 5 min at 47C. 2.3.2 Conventional PCR The two primer sets of duplex PCR, which were used to determine the cultured SARS-CoV and the samples of the clinically diagnosed SARS patients, were 5’-TAG GATTGCCTACGCAGACT-3’ and 5’-AGAGCCATGCCTAA CATGCT-3’ (for the 240 bp product), and 5’-ATTGGCT GTAACAGCTTGAC-3’ and 5’-TAGGGTAACCATTGACTT GG-3’ (for the 438 bp product), respectively. The duplex PCR mixture contained 2 mL of the reverse-transcribed product, 5 mL 106PCR buffer II, 0.5 mL LA Taq DNA polymerase, 1 mL (0.5 mL each of primers) duplex primers (20 mM), 5 mL Mg21 (25 mmol/L); sterile water was added to a total volume of 50 mL. The following PCR reaction protocol was employed: 947C for 5 min followed by 30 cycles at 947C for 30 s, 557C for 30 s, 727C for 30 s, and then 727C for 7 min. The products of PCR reaction were analyzed by visualizing with ethidium bromide staining. The reaction was finished on the GeneAmp PCR system 2400 (Perkin-Elmer). 2.4 Microchip fabrication The glass microchips were fabricated using standard photolithographic and wet chemical techniques as described elsewhere [16]. The microchannel design in Fig. 1 was transferred onto the substrates using a positive photoresist, photomask, and UV exposure. The microchannels were etched into the substrate in a dilute, stirred ? 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Figure 1. (a) Layout of the microfabricated channels; (b) PCR chip. The reactive area includes the reactive pool and the serpent-shape channel. The point of fluorescence detection is marked with an arrow. Potential of sample injection, 400 V/cm at the sample waste; grounding the sample reservoir for 30 s. The buffer and waste reservoirs had no potentials applied. Potentials of separation, 0, 1.1, 0.3, and 0.3 kV at the buffer, waste, sample, and sample waste reservoirs, respectively. HF/NH4F bath. To form the closed network of channels, a cover plate was bonded to the substrate over the etched channels by hydrolyzing the surfaces, bringing them into contact with each other, then processing thermally to 2007C in the vacuum oven for 1 h. Subsequently, the pair glass plates were placed between two pieces of smooth China plates and annealed in a Model SX3-4-10 programmable furnace (Zhonghuan Test Electrical Furance, Tianjin City, China). The temperature program was as follows: initial heating to 1007C for about 1 h, and to 6207C for about 3 h, followed by natural cooling of the furnace to room temperature. The channels were typically 20 mm deep and 50–60 mm wide at half-depth. The separation length was 3.0–3.5 cm. For forming reservoirs connected to the microchannels, the 2 mm diameter holes in the top glass plate (either substrate or cover plate) were drilled by the CK-250L ultrasound driller (Shantou Goldstar Ultrasonic Machinery, China). The typical volume of the reservoirs was 15 mL. Electrophoresis 2004, 25, 3032–3039 SARS-coronavirus detection on a microfluidic chip system 2.5 Microchip electrophoresis 3035 The electroosmotic flow in the channels was minimized using PVP-TBE buffer as a dynamic coating [17]. The channels were filled with a sieving buffer solution for microchip gel electrophoresis after they were oxidized in boiling H2SO4/H2O2 (1:1) for 10–15 min, followed by washing with deionized H2O. The coating was stable for multiple injection/separation cycles and provided reproducible separation performance. For DNA fragment sizing, 2.5% PVP solution in a 100 mM Tris/100 mM boric acid/2 mM EDTA buffer was used. An intercalating dye, SYTOX Orange nucleic acid stain, was added to the polymer solution at 1 mM for the PCR on-chip experiments. A DNA marker containing 11 DNA fragments of 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, and 1500 bp at approximately equal weight/volume concentration was used for PCR verification, product size determination, and estimations of the amplification efficiency. For the laser-induced fluorescence (LIF) detection, the LIF microfluidic chip analyzer was used. For the conventional injection/separation cycle, the sample was first loaded into the sample reservoir and then injected by applying 400 V/cm at the sample waste and grounding the sample reservoir for 30 s. The buffer and waste reservoirs had no potentials applied since the low diffusion coefficients of DNA fragments in the polymer sieving medium resulted in no substantial increase in the injection plug length or deterioration of separation channel. The relative potentials were switched to 0, 1.1, 0.3, and 0.3 kV at the buffer, waste, sample, and sample waste reservoirs, respectively. 1 inch, output power of 30 W). Aluminum cooling flakes are attached to the backside of the Peltier devices as heat sinks. A small cooling fan was also attached to each of the two aluminum blocks for convective cooling. During PCR thermal cycling, the 2/3 area of the entire chip was sandwiched between the two Peltier elements. The device temperature and Peltier surface temperature were monitored using thermocouples. Software inhouse made was used for thermal cycle temperature control. A drop of mineral oil on the bottom Peltier element ensured good thermal contact with the cycled region of the chip. All microchannels and reservoirs, excluding the PCR reaction area (including sample reservoir) in Fig. 1, were filled with sieving solution. The reaction well was first treated with 2.5 g/L bovine serum albumin (BSA) solution for 2–3 min to modify the inner glass surface of the milled reservoir. The BSA was then removed from the well and replaced with 3.5–4 mL of the PCR reaction mixture (individual or duplex PCR reaction mixture and cDNA of the sample). To prevent evaporation, all wells were topped with 6–7 mL of mineral oil. The temperature steps were 947C for 2 min, followed by 94, 55, and 727C with holding times of 45, 30, and 30 s for 30 cycles, and then 727C for 2 min to perform DNA amplification. The top thermoelectric element of the thermal cycler for the micro-PCR device was not placed in direct contact with the chip in order to eliminate the requirement of sealing the top reaction reservoir against contact-driven capillary wicking. Thus, the top assembly was positioned over the chip using 0.5 mm thick graphite strips as spacers. 2.6 Thermal cycling on-chip 2.7 Detection system Figure 2a shows a picture of the thermal cycler for the micro-PCR device, which was designed and built inhouse using two Peltier thermoelectric devices (1 inch6 Figure 3 shows schematically the layout of a micro-CE device with integrated confocal laser fluorescence detector and automicromanipulation stage. The dimensions Figure 2. (a) Thermal cycler for the micro-PCR device (home-made); (b) temperature cycling trace curve of the PCR chip. ? 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 3036 X. Zhou et al. Electrophoresis 2004, 25, 3032–3039 Figure 3. Schematic representation of the experimental instrument and the laser confocal detection arrangement. of the instrument were 50650645 cm. The output radiation (532 nm) from an air-cooled laser diode (LD)pumped solid-state laser (20 mW; Mektec Seiwa Corporation, Beijing, China) passes through a 532 nm filter (Omega Optical, Brattleboro, VT, USA). The beam is reflected by a dichroic beam-splitter (Omega Optical) and focused into the channel through a 206 microscope objective (0.4 N.A.). The emission signal is collected by the same objective and transmitted back through the dichroic beam-splitter. The emission beam passes through a bandpass filter (Omega Optical), which may be alternated easily to fit a wide selection of dyes, and is focused by a focusing lens through a 400 mm pinhole. The photomultiplier tube (R212; Japan) is mounted in an integrated detection module including high-voltage power supply, voltage divider, and amplifier. The chargecoupled device (CCD) camera was fixed at the same board as the photomultiplier tube in order to focus and observe the channel. The whole optical system was installed on the X-Y-Z translational stage (3-D micro- ? 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim manipulator, adjusting precision 1 mm), and the focus can be controlled via the picture displayed on the screen. 3 Results and discussion 3.1 Detection limit of the detection system The detection limit of Rhodamine 6G dye for the detection system with the home-made chip and a 100 mM TBE buffer is 6.67610213mol/L (S/N . 3). The linear range of detection was 4.0261026mol/L to 4.0261029mol/L (r = 0.9996). For detailed information we refer to the literature [18]. A 50 ng/mL F X-174/HaeIII digest DNA marker, with restriction fragments ranging in size from 72 to 1353 bp (72, 118, 194, 234, 271, 281, 310, 603, 872, 1078, 1353 bp), was serially diluted and determined on the detection system. When the S/N of the 603 bp fragment was 3, its corresponding concentration was Electrophoresis 2004, 25, 3032–3039 SARS-coronavirus detection on a microfluidic chip system 3037 0.2 ng/mL (the mass of the 603 bp fragment detected was 3.36 fg according to the injection volume). These data show that this detection system has a high sensitivity, which is over 100-fold higher than that of agarose gel electrophoresis in the conventional PCR assay [18]. The high assay sensitivity is important for early diagnosis of the diseases and early identifying of the suspected patients with SARS. 3.2 Design and optimization of SARS-CoV RT-PCR assay kit Primers were designed according to the conserved regions of open reading frame 1b (replicase 1B) following the Tor 2 SARS genome sequence in order to amplify sequences within two regions of SARS genome, 15240– 15612 and 17743–18349. Multiplex PCR is a modification of the basic PCR method, where multiple pairs of primers are used in the same reaction [19, 20]. A multiplex protocol appears to be useful for the investigation of different target sequences at the same time. By RT to cDNA and subsequent amplification, the RNA template can also be amplified by multiplex PCR. The SARS RNA genome is apt to mutation, which may lead to primers ill-matched with templates. Multiplex PCR, which can effectively reduce the possibility of false-negative PCR results caused by genome instability, amplifies different regions at the same time. So we designed nine pairs of primers according to the Tor 2 SARS genome sequence, and 2 of 9 pairs of primers were optimized as the primers for RT-PCR SARS-CoV assay kit. The sequences of two pairs of the primers are 5’-TAGGATTGCCTACGCAGACT-3’ and 5’-AGAGCCATGCCTAACATGCT-3’ (for the 240 bp product), and 5’-ATTGGCTGTAACAGCTTGAC-3’ and 5’-TAG GGTAACCATTGACTTGG-3’ (for the 438 bp product), respectively. The concentration of Mg21, enzymes, reaction time, cycling times, etc., for RT-PCR SARS-CoV assay kit were optimized. Optimization of RT-PCR SARS-CoV assay with SARS-CoV from the cultured Vero-E6 cells was finished by the GeneAmp PCR system 2400 (Perkin-Elmer) thermocycler. Reaction products from duplex PCR were analyzed by agarose gel electrophoresis (Fig. 4). 3.3 PCR-CE on-line A series of thermal transfer experiments have been conducted using different thicknesses of the glass (e.g., 2 mm, 0.5 mm, 0.15 mm), and it was found that the intrareservoir temperature would fit the set point temperature better by using a thinner glass cover (data not shown). However, the thinnest glass cover plate of the bottom of every reservoir is easily shattered, particularly during installation of the electrodes. Therefore, the thickness of the ? 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Figure 4. Agarose gel electropherogram of the products from duplex PCR. glass cover plate was kept thin (0.5 mm) in order to achieve a reasonable thermal cycle time. The temperature cycling of the PCR chip in a dual Peltier assembly is represented by trace curve in Fig. 2b, which shows the temperature profile for five cycles of the program inhouse software. The Peltier surface temperature and intra-reservoir temperature were measured by thermocouples. A built calibration chip contained an embedded thermocouple to assess the intra-reservoir temperature and compare it with the temperature of the Peltier surface. These data were used to design the thermal cycle profile of amplification experiments. The chips used in the experiments did not have the embedded thermocouple. Cycle times were initiated according to the intrareservoir temperature. Successful DNA amplification is dependent on the preliminary treatment of PCR reservoirs as well as on the reaction mixture volume. The pretreatment of PCR reservoirs was performed as described by Khandurina et al. [15]. Optimum thermal uniformity during heating/cooling cycles resulting in a good PCR efficiency was achieved using 3–4 mL PCR cocktail in 2 mm diameter reaction reservoirs and a serpent-shape channel. To achieve PCR amplification, a pair of PCR primers was first performed on the chip thermal cycler, and then the duplex PCR was manipulated. Figure 5a shows the electropherogram of 240 and 438 bp PCR products of the positive control of SARS-CoV. Amplification and electrophoretic analysis were on the same chip. The total analysis time was approximately 50–60 min including 30 thermal cycles and electrokinetic injection/separation manipulation. Figure 5b shows the electropherogram of the negative control (parainfluenza virus) and Fig. 5c the electropherogram of the 240 and 438 bp PCR products of the cultured SARSCoV. To identify the length of unknown amplified products or verify the sizes of known target sequences, PCR prod- 3038 X. Zhou et al. Electrophoresis 2004, 25, 3032–3039 Figure 5. Microfluidic chip electropherograms. (a) Positive control; (b) negative control; (c) cultured SARS-CoV (240 and 438 bp PCR products); (d) PCR product 1 100 bp DNA marker. ucts were mixed and coelectrophoresed with size markers of DNA as demonstrated in Fig. 5d for 240 and 438 bp products. The latter profile was obtained by simply adding a DNA marker solution to the sample reservoir containing the PCR product after mixing. Compared to the standard curve of the migration time vs. the fragment size of DNA marker, the size of the amplified DNA fragments was determined by their migration time. PCR with cDNA of the sample, the PCR chip was translated on the detection system to separate the PCR products. The chip-PCR and on-line CE separation for each sample were therefore performed on the chip (Fig. 1). Seventeen positives were achieved from 18 of the nasopharyngeal swabs from the clinically diagnosed patients with SARS by the microfluidic chip system. However, Fig. 6 shows that 12 positives of 18 SARS samples noted above were detected by the conventional PCR with agarose gel electrophoresis. 3.4 Determination of the clinical specimens All RNA of SARS-CoVs was extracted from 18 clinical SARS samples with RNAeasy Mini Kit (Qiagen) before performing the RT-PCR reaction. The RT reactions with the extracted RNA were performed on a conventional PCR cycler to get the cDNA of the sample. After chip- ? 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 4 Concluding remarks A microfluidic chip system was developed and successfully used in determining the SARS-CoV of specimens from clinically diagnosed SARS patients. The positive Electrophoresis 2004, 25, 3032–3039 SARS-coronavirus detection on a microfluidic chip system 3039 5 References Figure 6. Gel electropherogram of the positive products of RT-duplex PCR for the nasopharyngeal swabs of 18 clinically diagnosed patients with SARS. rate of the SARS-CoV for clinical SARS samples is up to 94.44% (17/18) using this system. The positive rate achieved by the conventional RT-PCR with agarose gel electrophoresis system was only 66.67% (12/18). Compared to the conventional system, the microfluidic chip system showed a higher positive rate (.27%), shorter testing time, and the potential application of determining the SARS-CoV of specimens. The authors thank the First Military Medical University for providing samples of clinically diagnosed SARS patients. This research was sponsored by the National Science Council of the People’s Republic of China under contract Nos. 20275039, 20035010, and 20299030, and in part by the Chinese Academy of Sciences for building the microfluidic chip analyzer and by the Dalian Institute of Chemical Physics, Chinese Academy of Sciences for studying SARS. Received December 30, 2003 ? 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim [1] WHO Summary table of SARS cases by country, 1 November 2002–7 August 2003, www.who.int/csr/sars/country/ 2003_08_15/en/. [2] SARS: Laboratory diagnosis tests. 29 April 2003, http:// www.who.int/csr/sars/diagnostictests/en. [3] McIntosh, K., Clin. Chem. 2003, 49, 845–846. [4] Hoard, J. C., Schultz, T. F., in: Collins, M., Clifton, N. J. (Eds.), Methods in Molecular Biology, Humana Press, Totowa, NJ 1991, Vol. 8. [5] Poon, L. L. M., Wong, O. K., Luk, W., Yuen, K. Y., Peiris, J. S. M., Guan, Y., Clin. Chem. 2003, 49, 845–846. [6] Wu, X. W., Cheng, G., Di, B., Yin, A. H., He, Y. S., Wang, M., Zhou, X.Y., He, L. J., Luo, K., Du, L., Chin. Med. J. 2003, 116, 988–990. [7] Freemantal, M., Chem. Eng. News 1999, 22, 27–36. [8] Northrup, M. A., Benett, B., Hadley, D., Long, G., Landre, P., Lehew, S., Richards, J., Stratton, P., Anal. Chem. 1998, 70, 918–992. [9] Belgrader, P., Benett, W., Hadley, D., Long, G., Mariella, R., Milanovich, F., Nelson, W., Richards, J., Stratton, P., Clin. Chem. 1998, 44, 2191–2194. [10] Belgrader, P., Benett, W., Hadley, D., Richards, J., Stratton, P., Mariella, R., Milanovich, F., Science 1999, 284, 449–450. [11] Ibrahim, M., Lofts, R., Jahrling, P., Henchal, E., Weedn, V., Northrup, M. A., Belgrader, P., Anal. Chem. 1998, 70, 2013– 2017. [12] Belgrader, P., Smith, J., Weedn, V., Northrup, M. A. J., Forensic Sci. 1998, 43, 315–319. [13] Effenhauser, C. S., Bruin, G. J. M., Paulus, A., Ehrat, M., Anal. Chem. 1997, 69, 3451–3457. [14] Yang, J., Liu, Y. J., Rauch, C. B., Stevens, R. H., Liu, R. L., Lenigk, R., Grodzinski, P., Lab Chip. 2002, 2, 179–187. [15] Khandurina, J., Mcknight, T. E., Jacobson, S. C., Waters, L. C., Foote, R. S., Ramsey, J. M., Anal. Chem. 2000, 72, 2995–3000. [16] Woolley, A. T., Mathies, R. A., Proc. Natl. Acad. Sci. USA 1994, 91, 11348. [17] Kopp, M. U., deMello, A. J., Manz, A., Science 1998, 280, 1046–1047. [18] Zhou, X. M., Dai, Z. P., Luo, Y., Liu, X., Wang, H., Lin, B. C., Chem. J. Chin. Univ. 2004, 3, 336–340. [19] Valassina, M., Cuppone, A. M., Cusi, M. G., Valensin, P. E., Clin. Diagn. Virol. 1997, 8, 227–232. [20] Cassinotti, P., Mietz, H., Siegl, G., J. Med. Virol. 1996, 50, 75–81. Final pg. 1 Final for! The exam is worth 60 points – roughly two problem sets. Ground Rules: 1. Unlike the problem sets, no collaboration with fellow students (or anyone else). 2. Unlike the problem sets, you should NOT use the internet to answer the questions (with the exception of one link provided in question #4). You may use any course material or any chemistry/biology textbook that is useful. 3. Please do not directly copy sentences from the attached paper or from the textbook in your answers. This is plagiarism. Write your answers in your own words. 4. Please feel free to reach out to me directly (hartley@ku.edu) if anything is ambiguous or you have questions. By signing/typing your name below, you are agreeing that you will follow the above ground rules. 1. (2 points) Final pg. 2 2. (9 points) Choose a video from your classmates’ videos on Blackboard that you have NOT already watched for the discussion (e.g., if you are in Group 1, choose a video from Group 5-8). Also, please choose a video on a topic different than your group’s topic. Watch the video and answer the following questions. For reference: Group 1: Atomic Absorption Spectroscopy Group 2: NMR Group 3: MRI Group 4: GC Group 5: GC-MS Group 6: Atomic Absorption Spectroscopy Group 7: NMR Group 8: GC a. Which group did you watch? (1 point) b. List three things you learned from the video. (3 points) c. In the research application discussed in the video, what question is being addressed by the featured instrumentation? (2 points) d. Identify another method that could be used to address the same question. List at least two advantages or disadvantages the alternative method has over the method in the video. (3 points) Final pg. 3 3. (10 points) Laser scanning confocal microscopy (LSCM) and scanning electron microscopy (SEM) are both surface methods that work by “raster” scanning, in which the sample is scanned line by line and data is collected at each point to generate an image. a. For each instrument, what is the source (primary beam) and what is detected (secondary beam)? (2 points) b. For each instrument, state whether the source is in direct line with the sample or at a 90° angle. Provide a short rationale for this instrumentation setup. (4 points) If your sample is a brain slice and you put it directly into either the LSM or SEM, very little signal would be detected. c. Explain why there is no signal for both methods. (2 points) d. Explain how you would prepare the sample to generate an observable signal for both methods. (2 points) Final pg. 4 4. (22 points) Plastic waste in the oceans is a major environmental problem. Contaminants that originate from plastics and are found in marine waters include phthalates. Phthalates represent a family of compounds with different modifications (R’s) on the ester groups. A common phthalate, diethylphthalate, is shown. In this experiment, your goal is to develop a quantitative assay to quantify phthalates in ocean water samples. First you will develop a method based on mass spectrometry. a. What type of ionization method and mass analyzer will you use for this method? (2 points) b. Why did you make the choices that you did in a? (1 point) c. Do you need to couple it to a separation method (GC or LC) to detect phthalates in ocean samples? (1 point) d. If c is yes, which one and why? If c is no, explain your answer. (1 point) Phthalates are typically ~1000 – 15,000 ng/L in ocean water. e. Choose a calibration method for your experiment (external curve, standard addition, or internal addition). (1 point) f. State which standard(s) you need for your chosen calibration method – be specific. (1 point) g. Your calibration curve should have at least 6 points. Please state what concentrations you will choose for your calibration curve points and if applicable, what concentration you will choose for an internal standard. (2 points) A mass spectrometry method involving fragmentation would be useful to identify phthalates that were previous not known to be in ocean water. h. Name two methods of mass spectrometry that involve compound fragmentation. (2 points) i. What is the difference between a product-ion spectra and a precursor-ion spectra? (2 points) j. Assume the phthalates fragment such that the R-groups from the esters are cleaved. Would a product-ion or precursor-ion scan be more useful for identifying previously unknown phthalates? (1 point) k. Explain your answer in j. (2 points) For the following alternative methods, state one advantage or one disadvantage (you choose which!) that the method would have compared to the method you developed above. Consider that you will be measuring the phthalates in a complex ocean water sample. Also, do not use the same answer twice (even if it applies to both)! l. Electrochemical method for detecting diethylphthalate (2 points) m. Capillary electrophoresis coupled to MS detection (2 points) n. Molecular sensor that emits fluorescence upon binding to diethylphthalate (2 points) Final pg. 5 5. (17 points) The attached paper uses the biological technique of PCR (polymerase chain reaction) with regards to a SARS-coronavirus – but not COVID-19 - as you will see this paper predates our current crisis. Although you do not need to understand the details of PCR to answer these questions, if you would like to know more about PCR while completing this exam, you can visit the tutorial at this website: https://www.khanacademy.org/science/ap-biology/gene-expression-andregulation/biotechnology/a/polymerase-chain-reaction-pcr A friendly reminder – please do not directly quote from the paper in your answer. This constitutes plagiarism. Please write your answers in your own words. a. What is the gap in technology being addressed by this paper? (2 points) b. What instrumental method(s) are being used to address the challenge? (2 points) c. What material and general fabrication methods were used to prepare the device? (2 points) d. How was the sample loaded onto the device? (1 point) e. The instrument diagram in Figure 3 has two major parts - one for reaction/separation and one for detection. Identify all modular components of both parts (i.e., sources, wavelength selectors, sample holders, transducers, and/or signal readout modules). (4 points) f. Which figures of merit are mentioned in the paper and what are the values? (3 points) g. Do the authors demonstrate that they have developed an improved technology? (1 point) h. Provide support for your answer in part g using data from the paper. (2 points) Micro- and Nanofabrication for Biomedical Research and Clinical Diagnostics Applications Ryan Grigsby Core Director 28 April 2021 Background • Microfabrication techniques were originally developed for the semiconductor and microelectronics industry • These techniques were adapted to miniaturize existing analytical systems, giving rise to new industries • Microelectromechanical systems (MEMS) • Microfluidics • Lab-on-a-Chip • Organ-on-a-Chip • Technological advancements over the past few decades have brought minimum feature sizes down to low nanometer scales Advantages of Miniaturization • Small samples sizes • Low cost • Device materials are relatively inexpensive • Low reagent use • Supplementary equipment is relatively inexpensive • Fast analysis times/High throughput • Low latency for real-time measurements • Closely approximates scale of biological systems • Amenable to point-of-care applications • Can be scaled for mass-production Patterning - Photolithography • Light source is typically UV • Hg-Arc: 365 nm (i), 405 nm (h), 436 nm (g) • Minimum feature size of about 1 micron • Deep UV techniques can get feature sizes down to about 40 nm. • Excimer laser sources • Immersion lithography • Extreme UV techniques can get feature sizes down to about 5 nm. • DUV techniques with Sequential patterning https://www.gregadunn.com/self-reflected/how-self-reflected-was-made/#1489201506375-41dfa123-9b27 Patterning - Direct Writing Electron Beam Lithography (EBL) and Direct Laser Writing (DLW) http://sam.zeloof.xyz/e-beam-lithography/ • EBL uses a modified scanning electron microscope • Resist is typically dissolved PMMA (plexi-glass) • Feature sizes down to about 5 nm https://www.knmf.kit.edu/DLW.php • DLW can be used to either create photomasks or to pattern resist films on substrates • Feature sizes down to about 600 nm Patterning - Direct Writing • • • • • • Focused Ion Beam (FIB) Milling A form of plasma etching where discreet structures can be made in the surface of a material without a mask Gallium is the most common plasma source for FIB Ga+ ions raster over the surface, similar to EBL, but no resist layer is required Frequently combined with SEM for imaging and process control FIB tools can include precursor gas for deposition Feature sizes down to about 15 nm Patterning - Micromachining • • • Computer Numerically Controlled (CNC) milling with high precision tools (0.5 µm positional accuracy), high spindle speeds (up to 60k r/m), and very small end mills (bits) Frequently used to make molds for hot embossing, but can also be used to direct-write into thermoplastics and elastomeric materials Structure height is limited by smallest end mill (3x dia.) milling bit 25 µm end mill substrate 80 µm hair Thin Films As A Process Tool • • • • • Etching Mask Lift-off Adhesion promotion Release agent General surface modification https://www.azonano.com/article.aspx?ArticleID=3219 https://www.allresist.com/wp-content/uploads/sites/2/2014/02/hmds.jpg Thin Film Deposition - Sputtering • • • General Types: DC, RF, reactive, ion beam Advantages • Low energy (< 1 kV, ~100 W/in2) • Can deposit at lower vacuum levels (mTorr) • High deposition rates (up to 75 nm/min) • Lower substrate temperatures (~150 °C) Disadvantages • Inefficient target usage • Difficult to sputter ferromagnetic materials • Conformal deposition • Limited source materials http://www.angstromsciences.com/sputtering-technology Thin Film Deposition - Evaporation • • General Types: Thermal and electron beam Advantages • Broad selection of available source materials, including some polymers • Efficient material usage • Line-of-sight coating Lower substrate temperatures (~150 °C) Disadvantages • High energy (up to 800 W for thermal; up to 40 kV for e-beam) • Sometimes requires ultra high vacuum (