A rapid and low-cost platform for detection of bacterial based on microchamber PCR microfluidic chip.

Polymerase chain reaction (PCR) has been considered as the gold standard for detecting nucleic acids. The simple PCR system is of great significance for medical applications in remote areas, especially for the developing countries. Herein, we proposed a low-cost self-assembled platform for microchamber PCR. The working principle is rotating the chamber PCR microfluidic chip between two heaters with fixed temperature to solve the problem of low temperature variation rate. The system consists of two temperature controllers, a screw slide rail, a chamber array microfluidic chip and a self-built software. Such a system can be constructed at a cost of about US$60. The micro chamber PCR can be finished by rotating the microfluidic chip between two heaters with fixed temperature. Results demonstrated that the sensitivity of the temperature controller is 0.1℃. The relative error of the duration for the microfluidic chip was 0.02 s. Finally, we successfully finished amplification of the target gene of Porphyromonas gingivalis in the chamber PCR microfluidic chip within 35 min and on-site detection of its PCR products by fluorescence. The chip consisted of 3200 cylindrical chambers. The volume of reagent in each volume is as low as 0.628 nL. This work provides an effective method to reduce the amplification time required for micro chamber PCR.

A portable instrument for on-site detection of heavy metal ions in water.

Based on differential pulse voltammetry technology, we developed a portable and affordable instrument for on-site detection of trace heavy metal pollutants in liquid through a disposable plastic pipette. It mainly consists of a six-electrode electrochemical sensor which is integrated in the instrument. The pipette chip is equipped with a pump valve, and thus, it can avoid contamination. We have analyzed the sensitivity and specificity of the electrochemical sensor for heavy metal detection. Experimental results demonstrated that the limit of detection for Pb, Hg, Cu, and Zn was 2.2 ng/mL, 2.5 ng/mL, 15.5 ng/mL, and 10 ng/mL, respectively. The limit of quantification for them was 10 ng/mL, 25 ng/mL, 25 ng/mL, and 14 ng/mL, respectively. The correlation coefficient between peak current and the target heavy metal concentration was above 0.96. Finally, we have tested the analytical performance of the self-build instrument by measuring heavy metal ions in industrial wastewater and rainwater, respectively. Such an instrument is user-friendly for all users even for the common people, and we can envision its wide application in future heavy metal pollutant detection in groundwater, tap water, and supernatant of soil solution.

Separation of subcellular fluorescent microspheres by capillary electrophoresis.

Fluorescent microspheres (FMs) are widely employed in diagnostics and life sciences research; here, we investigated the effect of capillary coating, polymer concentration, electric field strength, and sample concentration on the separation performance of 1.0 µm FMs in hydroxyethyl cellulose (HEC) by capillary electrophoresis (CE). Results showed that (1) capillary coating could enhance the fluorescence signal. (2) For HEC with the same molecular weight, the higher HEC concentration is, the later the first peak appears in the electropherogram. (3) When FMs are diluted, increasing the electric field strength can enhance the migration speed and reduce the aggregation of FMs. (4) The number of FMs calculated is close to the theoretical value when it is diluted 10,000 times. The optimum conditions for CE were as follows: 6 cm/8 cm of effective length and total length of the coated capillary, 0.3% HEC (1300 k), and 300 V/cm of electric field strength. Such a study is helpful for the development of a FM counting system. Graphical abstract.

Design and fabrication of portable continuous flow PCR microfluidic chip for DNA replication.

The reasons for restricting continuous flow polymerase chain reaction (CF-PCR) microfluidic chip from lab to application are that it is not portable and requires costly external precision pumps for sample injection. Herein, we employed water as the substitute for PCR solution, and investigated the effect of the cross-section, width-to-depth ratio, and the length ratio for three temperature zones of the micro channel on the thermal and flow distribution of fluid in micro tube by finite element analysis. Results show that the central velocity is uniform and stable velocity occupies the most if the cross-section is rectangular. The deviation between predefined temperature and theoretical temperature is slight and the fluid flux is the most if width-to-depth ratio is 1:1. It is suitable for the short DNA replication if the high temperature zone Wh is larger than the low temperature zone Wl, and vice versa. Then a portable CF-PCR microfluidic chip was fabricated and an automatic sample injection system was developed. As an application, we have successfully amplified the DNA of Treponema denticola in the chip within 8 min. Such a study may offer new insight into the design of CF-PCR microfluidic chip and promote it from lab-scale research to full-scale application.

Study of the peak broadening due to detection in the electrophoretic separation of DNA by CE and microchip CE and the application of image sensor for ultra-small detection cell length.

Narrow peaks are important to high-resolution and high-speed separation of DNA fragments by capillary electrophoresis and microchip capillary electrophoresis. Detection cell length is one of the broadening factors, which is often ignored in experiments. However, is it always safe to neglect detection cell length under any condition? To answer this question, we investigated the influence of detection cell length by simulation and experiments. A parameter named as detection cell length ratio was proposed to directly compare the detection cell length and the spatial length of sample band. Electrophoretic peaks generated by various detection cell length ratios were analyzed. A simple rule to evaluate the peak broadening due to detection cell length was obtained. The current states of the detection cell length of detection system and their reliabilities in capillary electrophoresis and microchip capillary electrophoresis were analyzed. Microchip capillary electrophoresis detection with an ultra-small detection cell length of 0.36 µm was easily achieved by using an image sensor.

The effect of electrophoretic parameters on separation performance of short DNA fragments.

Effective separation of short DNA fragments is important for the identification of PCR or LAMP products. We investigated the effect of electric field strength, sample plug width, effective length of the capillary, concentration and molecular weight of polymer on the separation performance of small DNA. Results demonstrated that the sample plug played a non-negligible role in the peak broadening. The migration time of DNA was exponentially decreased with the increase of electric field strength. Increasing effective length of capillary, concentration or molecular weight of HEC may improve the separation performance, but it was at the cost of long migration time.

Gene analysis of multiple oral bacteria by the polymerase chain reaction coupled with capillary polymer electrophoresis.

Capillary polymer electrophoresis is identified as a promising technology for the analysis of DNA from bacteria, virus and cell samples. In this paper, we propose an innovative capillary polymer electrophoresis protocol for the quantification of polymerase chain reaction products. The internal standard method was modified and applied to capillary polymer electrophoresis. The precision of our modified internal standard protocol was evaluated by measuring the relative standard deviation of intermediate capillary polymer electrophoresis experiments. Results showed that the relative standard deviation was reduced from 12.4-15.1 to 0.6-2.3%. Linear regression tests were also implemented to validate our protocol. The modified internal standard method showed good linearity and robust properties. Finally, the ease of our method was illustrated by analyzing a real clinical oral sample using a one-run capillary polymer electrophoresis experiment.

Analysis of small interfering RNA by capillary electrophoresis in hydroxyethylcellulose solutions.

The analysis of small interfering RNA (siRNA) is important for gene function studies and drug developments. We employed CE to study the separation of siRNA ladder marker, which were ten double-stranded RNA (dsRNA) fragments ranged from 20 to 1000 bp, in solutions of hydroxyethylcellulose (HEC) polymer with different concentrations and molecular weights (Mws). Migration mechanism of dsRNA during CE was studied by the mobility and resolution length (RL) plots. We found that the RL depended on not only the concentration of HEC, but also the Mw of HEC. For instance, RL of small dsRNA fragment was more influenced by concentration of high Mw HEC than large dsRNA fragment and RL of large dsRNA fragment was more influenced by concentration of low Mw HEC than small dsRNA fragment. In addition, we found electrophoretic evidence that the structure of dsRNA was more compact than dsDNA with the same length. In practice, we succeeded to separate the glyceraldehyde 3-phosphate dehydrogenase siRNA in the mixture of the siRNA ladder marker within 4 min.

Electrophoresis of periodontal pathogens in poly(ethyleneoxide) solutions with uncoated capillary.

Periodontitis is a prevalent inflammatory disease caused by different species of anaerobic bacteria such as Porphyromonas gingivalis (P.g), Treponema denticola (T.d), and Tannerella forsythia (T.f). We compared the separation result of DNA ladders in hydroxyethyl cellulose, poly(ethyleneoxide) (PEO), and polyethylene glycol and analyzed the effect of polymer concentration, electric field, and temperature of the background electrolyte on the separation performance. Results demonstrated that there was a linear relationship (R=0.942) for 100 to 700bp of DNA and its migration time. Finally, the polymerase chain reaction products of P.g, T.d, and T.f were successfully identified within 8.5 min in 0.5% PEO with uncoated capillary.

Optical microscopy imaging for the diagnosis of the pharmacological reaction of mouse embryonic stem cell-derived cardiomyocytes (mESC-CMs).

Quantitative diagnosis of pharmacological chronotropic reactions on mouse embryonic stem cell-derived cardiomyocytes (mESC-CMs) was successfully performed by utilizing derivative imaging analysis of videos recorded with a microscope camera at 30 Hz frame rate and 680 × 510 pixel resolution. The imaging analysis algorithm, developed in our lab, generated the contractile profile of the cells which was exploited for drug effect profiling. Six drugs such as isoproterenol (0.01-1 µM), quinidine (2-200 µM), propranolol (0.03-30 µM), verapamil (0.01-1 µM), sotalol (1-100 µM), and acetylsalicylic acid (0.1-10 µM) were administered and the quantitative medication effect was determined. Among the negative chronotropic agents administered, verapamil was found to be the most potent while sotalol was found to be the least potent at the micromolar level. Simultaneous measurement of the field potential and contractile motion in the verapamil effect test showed a coherent result. Moreover, this approach can provide insights into the contraction-relaxation conditions which are not available in the common electrophysiological approach. With these findings, it is expected that this study can aid in providing a simple and reliable in vitro mESC-CM-based screening platform for cardiovascular effect profiling of candidate drugs.

Synovial plicae and temporomandibular joint disorders: surgical findings.

PURPOSE: Synovial plicae and their relation to pain and disability have been reported in the orthopedic literature in association with the knee and other extremity joints. However, the occurrence of synovial plicae in the temporomandibular joint (TMJ) have rarely been reported. This report describes the surgical appearance, distribution, and histologic findings of synovial plicae in patients with TMJ recurrent dislocation and internal derangement. MATERIALS AND METHODS: Twenty consecutive patients, 16 with recurrent dislocation and 4 with internal derangement, who underwent open TMJ surgery by the same surgeon from 2010 to 2013 were studied retrospectively. RESULTS: Synovial plicae were detected in 18 of 28 joints (64.3%). Synovial plicae were observed in 15 of 24 joints (62.5%) with recurrent dislocation and in 3 of 4 joints (75%) with internal derangement. Histologic findings of these plicae were consistent with dense fibrous or cartilaginous tissues, with some exhibiting a synovial lining. CONCLUSIONS: Although the role of synovial plicae in TMJ disorders is unknown and unstudied, consideration should be given to investigating the possible relation of these structures to the signs and symptoms of TMJ disorders.

Determination and quantification of Escherichia coli by capillary electrophoresis.

Capillary electrophoresis (CE) is widely employed for the separation of nucleic acids or protein, but it is rarely applied in the quantification of Escherichia coli (E. coli). Here, we have analysed E. coli by CE with mercury lamp induced fluorescence, and demonstrated the relationship between its fluorescence intensity with the concentration of E. coli for the first time. The gradient concentration of E. coli was obtained by polymerase chain reaction (PCR) with different amplification cycles and dilution certain PCR products of E. coli, respectively. Results show that the peak area was linearly related to the logarithm of the concentration of E. coli and the logarithm of PCR replication numbers. The correlation coefficients R(2) are 0.957 and 0.966, respectively. The limit of detection (LOD) was found to be about 8.913 × 10(-15) mol µl(-1). The reproducibility of capillary electrophoresis may make this technique possible for quantitative measurement of bacteria in bio-analytical science.

High-throughput 3D microfluidic chip for generation of concentration gradients and mixture combinations.

Concentration gradient generation and mixed combinations of multiple solutions are of great value in the field of biomedical research. However, existing concentration gradient generators for single or two-drug solutions cannot simultaneously achieve multiple concentration gradient formations and mixed solution combinations. Furthermore, the whole system was huge, and required expensive auxiliary equipment, which may lead to complex operations. To address this problem, we devised a novel 3D microchannel network design, which is capable of creating all the desired mixture combinations and concentration gradients of given small amounts of the input solutions. As a proof of concept, the device we presented was verified by both colorimetric and fluorescence detection methods to test the efficiency. This can enable the implementation of one to three solutions with no driving pump and facilitate unique multiple types of more concentration gradients and mixture combinations in a single operation. We envision that this will be a promising candidate for the development of simplified methods for screening of the appropriate concentration and combination, such as various drug screening applications.

Direct count of fluorescent microspheres in a microfluidic chip based on the capillary electrophoresis method.

Fluorescent microspheres (FMs) are tiny particles with special functions that are widely employed in biological research. Counting of microscale FMs is a great challenge by capillary electrophoresis. Herein we developed a method to count 2 µm FMs based on a microfluidic chip with a gradual change in inner size. Such a microfluidic chip can inhibit sample blocking at the inlet of the capillary. The results showed that FMs migrated in the wide part of the microchannel side by side, and then passed through the narrow part one by one. There was a linear relationship between the number of peaks in the electropherogram and concentration of FMs if they were running in the microchannel for more than 20 min. A high separation voltage may lead to aggregation of FMs in the microchannels, and about 2 × 104 FMs can be counted within 30 min by this microfluidic chip.

Amplification of Escherichia coli in a Continuous-Flow-PCR Microfluidic Chip and Its Detection with a Capillary Electrophoresis System.

Polymerase chain reaction (PCR) is a traditional method employed for the amplification of a target gene that has played an important role in biomolecular diagnostics. However, traditional PCR is very time-consuming because of the low-temperature variation efficiency. This work proposes a continuous-flow-PCR (CF-PCR) system based on a microfluidic chip. The amplification time can be greatly reduced by running the PCR solution into a microchannel placed on heaters set at different temperatures. Moreover, as capillary electrophoresis (CE) is an ideal way to differentiate positive and false-positive PCR products, a CE system was built to achieve efficient separation of the DNA fragments. This paper describes the process of amplification of Escherichia coli (E. coli) by the CF-PCR system built in-house and the detection of the PCR products by CE. The results demonstrate that the target gene of E. coli was successfully amplified within 10 min, indicating that these two systems can be used for the rapid amplification and detection of nucleic acids.

Simultaneous amplification of DNA in a multiplex circular array shaped continuous flow PCR microfluidic chip for on-site detection of bacterial.

Based on time to place conversion, continuous flow polymerase chain reaction (CF-PCR) can realize a rapid amplification of DNA by running the PCR reagent in a serpentine microchannel but a larger space is required for each sample, which greatly reduces the efficiency of the CF-PCR. Herein, we propose a multiplex circular array shaped CF-PCR microfluidic chip for on-site detection of bacteria. There were 12 serpentine microchannels which were distributed on the disc in an annular form, and each microchannel consisted of an inlet for sample injection, and an outlet for the detection of the PCR products based on fluorescence. Samples could be simultaneously driven into each inlet by a one-to-twelve diverter through a syringe. Moreover, the method of adding fluorescent dyes at the end of the microchannel can solve the inhibition effect of excessive fluorescent dyes on the PCR reaction. The process finished with simultaneous amplification of 12 different target genes from Porphyromonas gingivalis, Treponema denticola, Tannerella forsythia, and Escherichia coli, and on-site detection of their corresponding positives within 23 min. The fastest detectable PCR reaction time was 5.38 ± 0.2 min at a flow rate of 1 mL h-1. For E. coli, the minimum detectable concentration was 2.5 × 10-3 ng µL-1 in this microfluidic system. Such a system can increase the throughput of CF-PCR for point-of-care testing of pathogens.

Lower fluidic resistance of double-layer droplet continuous flow PCR microfluidic chip for rapid detection of bacteria.

BACKGROUND: Rapid diagnosis of harmful microorganisms demonstrated its great importance for social health. Continuous flow PCR (CF-PCR) can realize rapid amplification of target genes by placing the microfluidic chip on heaters with different temperature. However, bubbles and evaporation always arise from heating, which makes the amplification not stable. Water-in-oil droplets running in CF-PCR microfluidic chip with uniform height takes long time because of the high resistance induced by long meandering microchannel. To overcome those drawbacks, we proposed a double-layer droplet CF-PCR microfluidic chip to reduce the fluidic resistance, and meanwhile nanoliter droplets were generated to minimize the bubbles and evaporation. RESULTS: Experiments showed that (1) fluidic resistance could be reduced with the increase of the height of the serpentine microchannel if the height of the T-junction part was certain. (2) Running speed, the size and the number of generated droplets were positively correlated with the cross-sectional area of the T-junction and water pressure. (3) Droplet fusion happened at higher water pressure if other experimental conditions were the same. (4) 0.032 nL droplet was created if the cross-sectional area of T-junction and water pressure were 1600 µm2 (40 × 40 µm) and 7 kPa, respectively. Finally, we successfully amplified the target genes of Porphyromonas gingivalis within 11'16″ and observed the fluorescence from droplets. SIGNIFICANCE AND NOVELTY: Such a microfluidic chip can effectively reduce the high resistance induced by long meandering microchannel, and greatly save time required for droplets CF-PCR. It offers a new way for the rapid detection of bacterial.

Novel gold-capped nanopillars imprinted on a polymer film for highly sensitive plasmonic biosensing.

Herein, a nanoporous alumina was fabricated to use as a mold in transforming nanopillar structures onto a thin film polymer by thermal nanoimprint lithography (NIL). The size of the pores was successfully controlled by varying the applied voltages and etching time. These nanoporous structures were transferred to the Cyclo-olefin polymer (COP) film surface from the porous mold by a thermal nanoimprinting process. A plasmonic substrate was fabricated by sputtering a thin layer of gold onto this nanopillar polymer structure, and the refractive index response in a variety of media was evaluated. Finally, the biosensing capacity of this novel plasmonic substrate was verified by analysis of Human immunoglobulin and achieved a minimum detection limit of 1.0 ng/mL. With the advantages of mass production with consistent reproducibility stemming from the nanoimprint fabrication process, our gold-capped polymeric pillars are ready for the transition from academic interest into commercialization systems for practical use in diagnostic applications.

Discrimination of primitive endoderm in embryoid bodies by Raman microspectroscopy.

Embryoid bodies (EBs), derived from aggregated embryonic stem (ES) cells, are capable of differentiating into all three germ layers, including the endoderm, mesoderm, and ectoderm. The initial stage of EB differentiation is the formation of a primitive endoderm (PE) layer located at the periphery of the aggregate. Raman microspectroscopy was employed to segregate PE cells from undifferentiated ES cells. The Raman spectra of the PE cells of the periphery of EBs, formed upon the withdrawal of leukemia inhibitory factor (LIF), were compared with those of the undifferentiated ES cells of the core of cell aggregates, formed in the presence of LIF. It was noticed that the PE cells have high contents of proteins and low contents of nucleic acids, lipids, and carbohydrates compared with ES cells. Also, we established the presence of another population of PE cells located in the core of the EBs. In addition, we identified some specific Raman markers to distinguish PE cells from ES cells (e.g., I(1003)/I(937)). This is the first study to investigate the PE cells of live EBs and define some Raman markers to distinguish them from undifferentiated ES cells.

A continuous flow PCR array microfluidic chip applied for simultaneous amplification of target genes of periodontal pathogens.

The concept of time to place conversion makes using a continuous flow polymerase chain reaction (CF-PCR) microfluidic chip an ideal way to reduce the time required for amplification of target genes; however, it also brings about low throughput amplicons. Although multiplex PCR can simultaneously amplify more than one target gene in the chip, it may easily induce false positives because of cross-reactions. To circumvent this problem, we herein fabricated a microfluidic system based on a CF-PCR array microfluidic chip. By dividing the chip into three parts, we successfully amplified target genes of Porphyromonas gingivalis (P.g), Tannerella forsythia (T.f) and Treponema denticola (T.d). The results demonstrated that the minimum amplification time required for P.g, T.d and T.f was 2'07'', 2'51'' and 5'32'', respectively. The target genes of P.g, T.d and T.f can be simultaneously amplified in less than 8'05''. Such a work may provide a clue to the development of a high throughput CF-PCR microfluidic system, which is crucial for point of care testing for simultaneous detection of various pathogens.