Polymerase Chain Reaction Chip Research Articles

Overcoming bubble formation in polydimethylsiloxane-made PCR chips: mechanism and elimination with a high-pressure liquid seal

The thermal expansion of gas and the air permeability of polydimethylsiloxane (PDMS) were previously thought to be the main causes of bubbles and water loss during polymerase chain reaction (PCR), resulting in a very complex chip design and operation. Here, by calculating and characterizing bubble formation, we discovered that water vapor is the main cause of bubbling. During PCR, heat increases the volume of the bubble by a factor of only ~0.2 in the absence of water vapor but by a factor of ~6.4 in the presence of water vapor. In addition, the phenomenon of “respiration” due to the repeated evaporation and condensation of water vapor accelerates the expansion of bubbles and the loss of water. A water seal above 109 kPa can effectively prevent bubbles in a bare PDMS chip with a simple structure, which is significant for the wide application of PDMS chips.

Ultrafast Metaphotonic PCR Chip with Near-Perfect Absorber.

Polymerase chain reaction (PCR) is the gold standard for nucleic acid amplification and quantification in diverse fields such as life sciences, global health, medicine, agricultural science, forensic science, and environmental science for global sustainability. However, implementing a cost-effective PCR remains challenging for rapid preventive medical action to the widespread pandemic diseases due to the absence of highly efficient and low-cost PCR chip-based POC molecular diagnostics. Here, this work reports an ultrafast metaphotonic PCR chip as a solution of a cost-effective and low-power-consumption POC device for the emerging global challenge of sustainable healthcare. This work designs a near-perfect photonic meta-absorber using ring-shaped titanium nitride to maximize the photothermal effect and realize rapid heating and cooling cycles during the PCR process. This work fabricates a large-area photonic meta-absorber on a 6-inch wafer cost-effectively using simple colloidal lithography. In addition, this work demonstrates 30 thermocycles from 65 (annealing temperature) to 95°C (denaturation temperature) within 3min 15s, achieving an average 16.66°Cs-1 heating rate and 7.77°Cs-1 cooling rate during thermocycling, succeeding rapid metaphotonic PCR. This work believes a metaphotonic PCR chip can be used to create a low-cost, ultrafast molecular diagnostic chip with a meta-absorber.

A high-performance digital polymerase chain reaction chip integrated with nanorods-decorated microchannel plate

Abstract In this paper, we report on a digital PCR chip integrated with microchannel plate (MCP) for the quantification of DNA molecules. MCP, a highly porous glass membrane, is employed here as microreactors. The density of the microreactors reaches up to 1600 mm−2 with a total number of 40,000 chambers each in 100 pL volumes embedded in a 5 × 5 mm2 MCP, which is 100 times larger than that of the conventional microfabricated chips. In addition, the MCP is functionalized with ZnO nanorods to enhance the fluorescence signal. The dynamic range is noted as 105 and the detection limit of λDNA is determined to be 1.4 copies/μL.

Photothermal-Based Multiplex Nested Digital PCR System for Rapid Detection of Foodborne Pathogens.

The rapid and sensitive detection of foodborne pathogens is crucial for ensuring food safety. Among virus testing methods, polymerase chain reaction (PCR) has served as the gold-standard technique in most food safety regulation organizations. However, to enhance the speed and efficiency of PCR, novel approaches are continually being explored. In this work, leveraging the photothermal effects and high thermal conductivity of gold nanoparticles, we have significantly improved the heating and cooling rates of thermal cycles, enabling ultra-fast PCR detection. Specifically, we present a pre-degassing multiplex digital PCR chip integrated with gold nanoparticles. We further developed a portable system with a light source for photothermal heating cycling, along with an optoelectronic sensor to analyze PCR amplification products after rapid thermal cycling. As proof of concept, the proposed chip and portable device was applied for the on-site detection of several types of foodborne pathogens, including Escherichia coli, Listeria monocytogenes, Staphylococcus aureus, and Salmonella. The whole system could distinguish those pathogens within 20 min, showing good potential for the rapid detection of multiple types of foodborne pathogens.

Designing Microfluidic PCR Chip Device Using CFD Software for the Detection of Malaria

Polymerase chain reaction (PCR) technique is one of the molecular methods in amplifying DNA for the detection of malaria. However, the collection and transportation of samples and the processing and dissemination of results via conventional PCR, especially when used for routine clinical practice, can hamper the technique’s sensitivity and specificity. The rampancy of such disease in the Philippines is aggravated by the limited supply of medical machinery and the poor economic state of the country; thus, the need to innovate a device for the early detection of malaria is necessary. With that, this study focuses on designing a microfluidic device that will mimic the function of a conventional genus-specific PCR based on the 18S rRNA gene to detect malaria parasites (Plasmodium falciparum) at low-grade parasitemia. The design was intended to be portable, accessible, and economical, which none from past literature has dealt with specifically for malaria detection. This in silico design is a first in the country specially crafted for such reasons. The proposed device was developed and simulated using ANSYS software for Computational Fluid Dynamics (CFD) analyses. The simulation shows that adding loops to the design increases its relative deviation but minimally compared to having only a straight path design. This indicates that looping is acceptable in designing a microfluidic device to minimize chip length. It was also found that increasing the cross-sectional area of the fluid path decreases the efficiency of the design. Lastly, among the three materials utilized, the chip made of polypropylene is the most efficient, with a relative deviation of 0.94 compared to polycarbonate and polydimethylsiloxane, which have relative deviations of 2.78 and 1.92, respectively. Future researchers may mesh the 44-cycle microfluidic chip due to the limitations of the software used in this study, and other materials, such as biocomposites, may be assessed to broaden the application of the design.

Non-invasive detection of tumor markers in salivary extracellular vesicles based on digital PCR chips

The World Health Organization (WHO) has stated that countless cancer patients could be saved if early detection and treatment were available. However, current clinical evaluation of tumors still relies primarily on imaging examinations and tissue biopsies. These methods not only require sophisticated equipment, but also have high false positive rates or invasive problems. Here, we describe a digital polymerase chain reaction (dPCR) chip for the detection of biomarkers in salivary extracellular vesicles (SEVs), which can be used to identify markers for the early diagnosis of tumors. Based on microfluidic technology fine microstructure and microfluidics operations, this dPCR chip can accurate quantitative SEVs in a variety of tumor markers, and shows extremely strong sensitivity (10 copies). In the detection of clinical samples, the chip can effectively distinguish lung cancer cases from normal controls (P < 0.001; two-tailed t-test), and in the detection of extremely low concentration samples, it shows considerably higher precise quantitative ability than quantitative real-time polymerase chain reaction (qPCR). Overall, this study may shed new light on non-invasive early screening of tumor markers by detecting extracellular vesicle-associated markers in saliva.

An integrated digital polymerase chain reaction chip for multiplexed meat adulteration detection.

Meat adulteration detection is a common concern of consumers. Here, we proposed a multiplex digital polymerase chain reaction method and a low-cost device for meat adulteration detection. Using a polydimethylsiloxane microfluidic device, polymerase chain reaction reagents could be pump-free loaded into microchambers (40×40 chambers) automatically. Due to the independence of multiplex fluorescence channels, deoxyribonucleic acid templates extracted from different animal species could be distinguished by one test. In this paper, we designed primers and probes for four types of meat (beef, chicken, pork, and duck) and labeled each of the four fluorescent markers (hexachlorocyclohexane [HEX], 6-carboxyfluorescein [FAM], X-rhodamine [ROX], and cyanine dyes 5 [CY5]) on the probes. Specific detection and mixed detection experiments were performed on four types of meat, realizing a limit of detection of 3 copies/µL. A mixture of four different species can be detected by four independent fluorescence channels. The quantitative capability of this method is found to meet the requirements of meat adulteration detections. This method has great potential for point-of-care testing together with portable microscopy equipment.

Ultrafast Nucleic Acid Detection Equipment with Silicon-Based Microfluidic Chip

Recently, infectious diseases, such as COVID-19, monkeypox, and Ebola, are plaguing human beings. Rapid and accurate diagnosis methods are required to preclude the spread of diseases. In this paper, an ultrafast polymerase chain reaction (PCR) equipment is designed to detect virus. The equipment consists of a silicon-based PCR chip, a thermocycling module, an optical detection module, and a control module. Silicon-based chip, with its thermal and fluid design, is used to improve detection efficiency. A thermoelectric cooler (TEC), together with a computer-controlled proportional-integral-derivative (PID) controller, is applied to accelerate the thermal cycle. A maximum of four samples can be tested simultaneously on the chip. Two kinds of fluorescent molecules can be detected by optical detection module. The equipment can detect viruses with 40 PCR amplification cycles in 5 min. The equipment is portable, easily operated, and low equipment cost, which shows great potential in epidemic prevention.

Simulation of Rapid Thermal Cycle for Ultra-Fast PCR.

The polymerase chain reaction (PCR) technology is a mainstream detection method used in medical diagnoses, environmental monitoring, food hygiene, and safety. However, the systematic analysis of a compact structure with fast temperature changes for an ultra-fast PCR device that is convenient for on-site detection still lacks investigation. To overcome the problems of low heating efficiency and non-portability of PCR devices currently used, a miniaturized PCR system based on a microfluidic chip, i.e., lab-on-chip technology, has been proposed. The main objective of this paper is to explore the feasibility of using a heat resistor that can reach a fast heating rate and temperature uniformity combined with air cooling technology for rapid cooling and to investigate the influences of various pattern designs and thicknesses of the resistor on heating rates and temperature uniformity. Additionally, a PCR chip made of various materials with different thermal properties, such as surface emissivity, thermal conductivity, mass density, and heat capacity at constant pressure is analyzed. In addition to the heat loss caused by the natural convection of air, the radiation loss of the simulation object is also considered, which makes the model much closer to the practical situation. Our research results provide a considerable reference for the design of the heating and cooling modules used in the ultra-fast PCR protocol, which has great potential in In Vitro Diagnosis (IVD) and the PCR detection of foodborne pathogens and bacteria.

A Portable Continuous-Flow Polymerase Chain Reaction Chip Device Integrated with Arduino Boards for Detecting Colla corii asini.

Food security is a significant issue in modern society. Because morphological characters are not reliable enough to distinguish authentic traditional Chinese medicines, it is essential to establish an effective and applicable method to identify them to protect people’s health. Due to the expensive cost of the manufacturing process and the large volume of the analytical system, the need to build a portable and cheap device is urgent. This work describes the development of a portable nucleic acid amplification device integrated with thermal control and liquid pumping connecting to Arduino boards. We present a novel microfluidic polymerase chain reaction (PCR) chip with symmetric isothermal zones. The total chip volume is small, and only one Arduino board is needed for thermal control. We assemble a miniaturized liquid pump and program an Arduino file to push the sample mixture into the chip to implement the PCR process. In the proposed operation, the Nusselt number of the sample flow is less than one, and the heat transfer is conduction only. Then we can ensure temperature uniformity in specific reaction regions. A Colla corii asini DNA segment of 200 bp is amplified to evaluate the PCR performance under the various operational parameters. The initial concentration for accomplishing the PCR process is at least 20 ng/μL at the flow rate of 0.4 μL/min in the portable continuous flow PCR (CFPCR) device. To our knowledge, our group is the first to introduce Arduino boards into the heat control and sample pumping modules for a CFPCR device.

MRG Chip: A High-Throughput qPCR-Based Tool for Assessment of the Heavy Metal(loid) Resistome.

Bacterial metal detoxification mechanisms have been well studied for centuries in pure culture systems. However, profiling metal resistance determinants at the community level is still a challenge due to the lack of comprehensive and reliable quantification tools. Here, a novel high-throughput quantitative polymerase chain reaction (HT-qPCR) chip, termed the metal resistance gene (MRG) chip, has been developed for the quantification of genes involved in the homeostasis of 9 metals. The MRG chip contains 77 newly designed degenerate primer sets and 9 published primer sets covering 56 metal resistance genes. Computational evaluation of the taxonomic coverage indicated that the MRG chip had a broad coverage matching 2 kingdoms, 29 phyla, 64 classes, 130 orders, 226 families, and 382 genera. Temperature gradient PCR and HT-qPCR verified that 57 °C was the optimal annealing temperature, with amplification efficiencies of over 94% primer sets achieving 80-110%, with R2 > 0.993. Both computational evaluation and the melting curve analysis of HT-qPCR validated a high specificity. The MRG chip has been successfully applied to characterize the distribution of diverse metal resistance determinants in natural and human-related environments, confirming its wide scope of application. Collectively, the MRG chip is a powerful and efficient high-throughput quantification tool for exploring the microbial metal resistome.

Portable Heating System Based on a Liquid Metal Bath for Rapid PCR

With the outbreak of COVID-19 around the world, rapid and accurate detection of new coronaviruses is the key to stop the transmission of the disease and prevent and control the novel coronavirus, among which polymerase chain reaction (PCR) is the mainstream nucleic acid detection method. A temperature cycling device is the core of the PCR amplification micro-device. The precision of the temperature control and temperature change rate directly affect the efficiency of PCR amplification. This study proposes a new PCR method based on rapid PCR chip optimization of a liquid metal bath, which realizes precise and rapid temperature rise and fall control. We systematically explored the feasibility of using liquid metals with different melting points in the system and proposed a 47 °C bismuth-based liquid metal bath as the heat conduction medium of the system to optimize the system. The heat conduction properties of the thermally conductive silicone oil bath were compared. Compared with the thermally conductive silicone oil bath, thermal cycle efficiency is improved nearly 3 times. The average heating rate of the liquid metal bath is fast, and the temperature control stability is good, which can significantly reduce the hysteresis, and the temperature change curve is more gentle, which can greatly improve the efficiency of PCR amplification. The results of gene amplification using rat DNA as the template and SEC61A as the target also indicate that the system can be successfully used in PCR devices, and the types of PCR containers can be not limited to PCR tubes. Based on the experiment, we proved that the PCR method optimized by the liquid metal bath multi-gene rapid PCR chip can further improve the temperature response speed. It has the advantages of accurate data, fast response speed, low price, safety, and environmental protection and can effectively reduce the time of PCR and improve the application efficiency. As far as we know, this is the first international report on using a liquid metal bath to do rapid-cooling PCR.

Ultrafast DNA Amplification Using Microchannel Flow-Through PCR Device.

Polymerase chain reaction (PCR) is limited by the long reaction time for point-of-care. Currently, commercial benchtop rapid PCR requires 30–40 min, and this time is limited by the absence of rapid and stable heating and cooling platforms rather than the biochemical reaction kinetics. This study develops an ultrafast PCR (<3 min) platform using flow-through microchannel chips. An actin gene amplicon with a length of 151 base-pairs in the whole genome was used to verify the ultrafast PCR microfluidic chip. The results demonstrated that the channel of 56 μm height can provide fast heat conduction and the channel length should not be short. Under certain denaturation and annealing/extension times, a short channel design will cause the sample to drive slowly in the microchannel with insufficient pressure in the channel, causing the fluid to generate bubbles in the high-temperature zone and subsequently destabilizing the flow. The chips used in the experiment can complete 40 thermal cycles within 160 s through a design with the 56 µm channel height and with each thermal circle measuring 4 cm long. The calculation shows that the DNA extension speed is ~60 base-pairs/s, which is consistent with the theoretical speed of the Klen Taq extension used, and the detection limit can reach 67 copies. The heat transfer time of the reagent on this platform is very short. The simple chip design and fabrication are suitable for the development of commercial ultrafast PCR chips.

Wet-Etched Microchamber Array Digital PCR Chip for SARS-CoV-2 Virus and Ultra-Early Stage Lung Cancer Quantitative Detection.

We report a novel design of chamber-based digital polymerase chain reaction (cdPCR) chip structure. Using a wet etching process and silicon-glass bonding, the chamber size can be adjusted independently of the process and more feasibly in a normal lab. In addition, the structure of the chip is optimized through hydrodynamic computer simulations to eliminate dead space when the sample is injected into the chip. The samples will be distributed to each separated microchambers for an isolated reaction based on Poisson distribution. Due to the difference in expansion coefficients, isolation of the sample in the microchambers by the oil phase on top ensures homogeneity and independence of the sample in the microchambers. The prepared microarray cdPCR chip enables high-throughput and high-sensitivity quantitative measurement of the SARS-CoV-2 virus gene and the mutant lung cancer gene. We applied the chip for the detection of different concentrations of the mix containing the open reading frame 1ab (ORF1ab) gene, the most specific and conservative gene region of the SARS-CoV-2 virus. In addition to this, we also successfully detected the fluorescence of the epidermal growth factor receptor (EGFR) mutant gene in independent microchambers. At a throughput of 46 200 microchambers, solution mixtures containing both genes were successfully tested quantitatively, with a detection limit of 10 copies/μL. Importantly, the chips are individually inexpensive and easy to industrialize. In addition, the microarray can provide a unified solution for other viral sequences, cancer marker assay development, and point-of-care testing (POCT).

Temperature non-uniformity detection on dPCR chips and temperature sensor calibration.

A microfluidic-based digital polymerase chain reaction (dPCR) chip requires precise temperature control as well as uniform temperature distribution to ensure PCR efficiency. However, measuring local temperature and its distribution over thousands of μL/nL-volume samples with minimum disturbance is challenging. Here, we present a method of non-contact localized temperature measurement for determination of the non-uniformity of temperature distribution over a dPCR chip. We filled the dPCR chip with a PCR solution containing amplified DNA fragments with a known melting temperature (TM). We then captured fluorescent images of the chip when it was heated from 70 to 99 °C, plotted the fluorescence intensity of each partition as a function of temperature, and calculated measured TM values from each partition. Finally, we created a 3-D map of the dPCR chip with the measured TM as the parameter. Even when the actual TM of the PCR solution was constant, the measured TM value varied between locations due to temperature non-uniformity in the dPCR chip. The method described here thereby characterized the distribution of temperature non-uniformity using a PCR solution with known TM as a temperature sensor. Among the non-contact temperature measurement methods, the proposed TM-based method can determine the temperature distribution within the chip, instead of only at the chip surface. The method also does not suffer from the undesirable photobleaching effect of fluorescein-based temperature measurement method. Temperature determination over the dPCR chip based on TM allowed us to calibrate the temperature sensor and improve the dPCR configuration and precision. This method is also suitable for determining the temperature uniformity of other microarray systems where there is no physical access to the system and thus direct temperature measurement is not possible.

A plasmonic gold nanofilm-based microfluidic chip for rapid and inexpensive droplet-based photonic PCR

Polymerase chain reaction (PCR) is a powerful tool for nucleic acid amplification and quantification. However, long thermocycling time is a major limitation of the commercial PCR devices in the point-of-care (POC). Herein, we have developed a rapid droplet-based photonic PCR (dpPCR) system, including a gold (Au) nanofilm-based microfluidic chip and a plasmonic photothermal cycler. The chip is fabricated by adding mineral oil to uncured polydimethylsiloxane (PDMS) to suppress droplet evaporation in PDMS microfluidic chips during PCR thermocycling. A PDMS to gold bonding technique using a double-sided adhesive tape is applied to enhance the bonding strength between the oil-added PDMS and the gold nanofilm. Moreover, the gold nanofilm excited by two light-emitting diodes (LEDs) from the top and bottom sides of the chip provides fast heating of the PCR sample to 230 °C within 100 s. Such a design enables 30 thermal cycles from 60 to 95 °C within 13 min with the average heating and cooling rates of 7.37 ± 0.27 °C/s and 1.91 ± 0.03 °C/s, respectively. The experimental results demonstrate successful PCR amplification of the alcohol oxidase (AOX) gene using the rapid plasmonic photothermal cycler and exhibit the great performance of the microfluidic chip for droplet-based PCR.

Direct digital polymerase chain reaction chip for the detection of EGFR T790M mutation in plasma

Nucleic acid extraction and purification before amplification is considered an essential step for nucleic acid amplification testing. However, this may cause losses or introduce errors that can lead to inaccurate results, especially when using samples with a small nucleic acid concentration. Here, we developed a direct digital chip that enabled us to detect nucleic acid without DNA extraction and purification. We have developed a self-priming liquid-dispensing digital PCR chip that does not require any external power. This is a robust anti-evaporation digital PCR chip with fast sampling and accurate quantification performance. Using this chip, we have established an on-chip direct nucleic acid amplification method that does not require nucleic acid extraction and purification for liquid biopsy samples. In order to verify the feasibility of this chip for clinical samples, we detected the EGFR T790M mutation from plasma. Results showed that EGFR T790M mutation could be detected with an accuracy of 100% and a sensitivity of 0.01%. Without nucleic acid extraction and purification, the assay avoids complex pre-processing, thus saving time and achieving precise quantification. We expect our direct digital PCR chip to have practical applications in diagnosis, screening, and research, especially in resource-deprived regions.

Effects of single and multiple transplantations of human umbilical cord mesenchymal stem cells on the recovery of ovarian function in the treatment of premature ovarian failure in mice

BackgroundCurrently, there is no effective treatment for premature ovarian failure (POF), and stem cell therapy is considered the most promising treatment. Human umbilical cord blood mesenchymal stem cells (hUC-MSCs) have shown good regenerative ability in various diseases, including POF; however, their underlying mechanism and dosage for POF treatment remain unclear. This study aimed to compare the effect of single and multiple injections of hUC-MSCs on ovarian function repair in chemotherapy-induced POF.MethodsFemale mice were intraperitoneally injected with 30 mg/kg busulfan and 120 mg/kg cyclophosphamide (CTX) to induce POF. In the single hUC-MSC injection group, hUC-MSCs were transplanted into mice D7 after CTX and busulfan administration, while in the multiple injection group, hUC-MSCs were transplanted on D7, D14, and D21 after CTX and busulfan administration. We evaluated the ovarian morphology, fertility, follicle-stimulating hormone and estradiol concentrations, follicle count, POF model, and cell transplantation results. In addition, real-time polymerase chain reaction, immunohistochemistry, and miRNA and mRNA chips were used to evaluate the effect of the cell therapy.ResultsOvary size, number of follicle at all developmental stages, and fertility were significantly reduced in the POF group compared with the control. Under hUC-MSC treatment, the ovarian morphology and follicle count were significantly restored, and fertility was significantly increased. By comparing the single and multiple hUC-MSC injection groups, we found that the anti-Müllerian hormone and Ki-67 levels were significantly increased in the multiple hUC-MSC group on D60 after chemotherapy. The expression of stimulating hormone receptors, inhibin α, and inhibin β was significantly restored, and the therapeutic effect was superior to that of the single hUC-MSC injection group.ConclusionThese results indicate that hUC-MSCs can restore the structure of injured ovarian tissue and its function in chemotherapy-induced POF mice and ameliorate fertility. Multiple hUC-MSC transplantations have a better effect on the recovery of ovarian function than single hUC-MSC transplantation in POF.

Ultrafast and Real-Time Nanoplasmonic On-Chip Polymerase Chain Reaction for Rapid and Quantitative Molecular Diagnostics.

Advent and fast spread of pandemic diseases draw worldwide attention to rapid, prompt, and accurate molecular diagnostics with technical development of ultrafast polymerase chain reaction (PCR). Microfluidic on-chip PCR platforms provide highly efficient and small-volume bioassay for point-of-care diagnostic applications. Here we report ultrafast, real-time, and on-chip nanoplasmonic PCR for rapid and quantitative molecular diagnostics at point-of-care level. The plasmofluidic PCR chip comprises glass nanopillar arrays with Au nanoislands and gas-permeable microfluidic channels, which contain reaction microchamber arrays, a precharged vacuum cell, and a vapor barrier. The on-chip configuration allows both spontaneous sample loading and microbubble-free PCR reaction during which the plasmonic nanopillar arrays result in ultrafast photothermal cycling. After rapid sample loading less than 3 min, two-step PCR results for 40 cycles show rapid amplification in 264 s for lambda-DNA, and 306 s for plasmids expressing SARS-CoV-2 envelope protein. In addition, the in situ cyclic real-time quantification of amplicons clearly demonstrates the amplification efficiencies of more than 91%. This PCR platform can provide rapid point-of-care molecular diagnostics in helping slow the fast-spreading pandemic.

Accomplishment of one-step specific PCR and evaluated SELEX process by a dual-microfluidic amplified system.

One of the main obstacles for systematic evolution of ligands by exponential enrichment (SELEX) failure is the generation of a non-specific product, as selection-inherent amplification procedures tend to form by-products, which prevents the enrichment of target-binding aptamers. Herein, we reported a dual-microfluidic amplified system (dual-MAS) based on the real-time polymerase chain reaction (PCR) detection chip and the large volume PCR chip for one-step specific PCR and for evaluating the SELEX process. First, it is a simple method to accomplish analytical PCR and amplification PCR in one step, and the optimal number of cycles for generating the specific PCR product is the cycles when the slope of the linear amplification period of the real-time PCR curve begins to decrease. Second, the time used by the dual-MAS for generating a specific PCR product is reduced to 30 min, and the multi-functional dual-MAS can simultaneously evaluate the SELEX process by providing important information on the amounts of enriched sequences and the library diversity in every round of SELEX. In addition, pollution contamination and fragment loss can be significantly avoided in the closed chip. Last, the specific PCR product, the amounts of enriched sequences, and the library diversity can be obtained for every single SELEX in just 30 min. Compared with current methods, this system can reduce the time for generating a specific PCR product and SELEX, and it is easier to choose the optimal number of cycles for a specific PCR product. In a word, it is a sensitive, simple, and rapid strategy to improve the specificity of the PCR product and make the process of SELEX in a controlled way.