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.

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.

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.

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.

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.

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.

Capillary electrophoresis of DNA with high resolution based on copoly(pentaerythritoltetra succinimidylcarboxypentyl/aminopropyl polyoxyethylene) hydrogel.

Capillary gel electrophoresis is widely applied for determination of sequence and size of DNA, in which the sieving gel plays an unignorable role. Herein, a pore-size controllable hydrogel was synthesized in the capillary with two symmetrical tetrahedron-like macromonomers consisting of pentaerythritoltetra (succinimidylcarboxypentyl) polyoxyethylene (PS) and pentaerythritoltetra (aminopropyl) polyoxyethylene) (PA). By capillary electrophoresis of the DNA fragments with this hydrogel, it is found that a homogenous structure of hydrogel which is more suitable for the DNA separation can be achieved when the molecular weight of PA is approximate to that of PS. DNA fragments smaller than 1500 bp can be well resolved in this hydrogel within 13 min. More than 100 consecutive runs can be carried out in such a dynamically coated capillary before performance begins to degrade. Notably, such hydrogel can realize separation of dsDNA up to single base pair resolution and same length of dsDNA with 1 bp difference.

A rapid nucleic acid concentration measurement system with large field of view for a droplet digital PCR microfluidic chip.

Droplet digital polymerase chain reaction (ddPCR) is an effective technique, with unparalleled sensitivity, for the absolute quantification of target nucleic acids. However, current commercial ddPCR devices for detecting the gene chip are time consuming due to complex image stitching. To address this issue, we propose a universal concentration determination system and realize one-time gene chip imaging with high resolution. All the functional units are controlled by self-developed software using the PyQt5 module in Python. Without stitching technology, images of the ddPCR chip (28 mm × 18 mm) containing 20 000 independent 0.81 nL micro chambers can be obtained in less than 15 seconds, which saves about 165 seconds. A white laser light source (2 mW cm-2) was employed as a substitute for the mercury lamp. Its wavelength matches well with typical fluorescent dyes (e.g., HEX, ROX and Cy5), and thus it can strengthen the fluorescence intensity for weak signals. The results also demonstrated that the correlation coefficient for the measured concentration and theoretical value was above 99%, by testing the ddPCR products with COVID-19 virus. Such a system can greatly reduce the time required for image acquisition and DNA concentration determination, and thus is able to speed up the lab-to-application process for ddPCR technology.

Multiplex amplification of target genes of periodontal pathogens in continuous flow PCR microfluidic chip.

Porphyromonas gingivalis (P.g), Treponema denticola (T.d), and Tannerella forsythia (T.f) are believed to be the major periodontal pathogens that cause gingivitis, which affects 50-90% of adults worldwide. Microfluidic chips based on continuous flow PCR (CF-PCR) are an ideal alternative to a traditional thermal cycler, because it can effectively reduce the time needed for temperature transformation. Herein, we explored multi-PCR of P.g, T.d and T.f using a CF-PCR microfluidic chip for the first time. Through a series of experiments, we obtained two optimal combinations of primers that are suitable for performing multi-PCR on these three periodontal pathogens, with amplicon sizes of (197 bp, 316 bp, 226 bp) and (197 bp, 316 bp, 641 bp), respectively. The results also demonstrated that by using multi-PCR, the amplification time can be reduced to as short as 3'48'' for the short-sized amplicons, while for T.f (641 bp), the minimum time required was 8'25''. This work provides an effective way to simultaneously amplify the target genes of P.g, T.d and T.f within a short time, and may promote CF-PCR as a practical tool for point-of-care testing of gingivitis.

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.

A SERS-based capillary sensor for the detection of mercury ions in environmental water.

Mercury ion (Hg2+) is one of the most toxic heavy metal ions which will cause permanent damage to the brain and kidneys. So, it is important to develop a sensitive, simple and reliable approach to detect Hg2+. In this work, we report a surface-enhanced Raman scatting (SERS) sensor by decorating the inner wall of capillary with 4,4'-dipyridyl (Dpy) functionalized silver nanoparticles (AgNPs). The main advantage of this sensor is that it can collect samples directly by capillary force and carry out on-site analysis by combining portable Raman spectrometer. In the presence of Hg2+, the Dpy molecules would be separated from the surface of AgNPs and coordinated with Hg2+, resulting in a decrease in the SERS signal. A linear correlation of Raman intensity with Hg2+ concentrations from 1 to 100 part-per-billion (ppb) was obtained for quantitative analysis and the limit of detection (LOD) was determined to be 0.1 ppb. The good reproducibility and selectivity of the sensor were also demonstrated. In addition, the sensors were successfully applied to detect Hg2+ in real environmental water samples, and the sampling process provided operation convenience compared to conventional methods. These results indicated that these capillary sensors had great potential for Hg2+ detection in practical use.

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.

Raman imaging diagnosis of the early stage differentiation of mouse embryonic stem cell (mESC).

Raman microspectroscopy as a non-invasive and label-free technique was applied to diagnose the early stage differentiation of mouse embryonic stem cells. The differentiated and undifferentiated embryonic bodies (EBs) were cultured using handing drop method by the control of Leukemia Inhibitory Factor (LIF). Raman spectra of the periphery cells of differentiated EBs (PrE cells) and those of the interior of undifferentiated EBs (ES cells) were obtained to diagnose the stem cells of different differentiation. It was found from the spectra that the protein content increased as the cells differentiated. Principal component analysis (PCA) was carried out to further analyze the differences between ES cells and PrE cells. The first three principle components contained 98.19% from the total variance. Characteristic bands of ES and PrE cells were chosen to acquire Raman images of two cells according to the results of PCA. In the Raman images, PrE cells had a clear and bright outline in the peripheral areas while ES cells were difficult to identify, this could be a distinct characteristic to discriminate them. The result of the Raman images was consistent with the biological agreement that the differentiated cells were distributed around the periphery.

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.

All-in-one microfluidic device for on-site diagnosis of pathogens based on an integrated continuous flow PCR and electrophoresis biochip.

Current continuous flow polymerase chain reaction (CF-PCR) microfluidic chips require external precision syringe pumps and off-line methods (e.g., electrophoresis and hybridization) to detect PCR products, resulting in complex operations and possible cross-contamination and consequently CF-PCR is still confined to laboratories. Herein, a portable all-in-one microfluidic device is fabricated for rapid diagnosis of pathogens based on an integrated CF-PCR and electrophoresis biochip. A new method was proposed for automatic sample injection into the chip which can substitute the costly external precision syringe pump. It not only achieves rapid DNA amplification and on-site PCR product detection, but also realizes automatic sample injection. As an application, three periodontal pathogens (e.g., Porphyromonas gingivalis, Treponema denticola and Tannerela forsythia) were successfully amplified in the device. Treponema denticola was amplified in as short as 2'31'', and detection of PCR products was completed within 3'43''. The minimum number of bacteria that can be amplified was 125 cfu per µl. The all-in-one device has the potential to be applied in point-of-care nucleic acid testing for diseases.

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.

Rapid and quantitative detection of trace Sudan black B in dyed black rice by surface-enhanced Raman spectroscopy (SERS).

The use of Sudan black B as coloring agent in foods is forbidden for its toxicology effect on human organs. This work proposes an efficient and sensitive method for food security inspection targeting Sudan black B. Surface-enhanced Raman spectroscopy (SERS) is applied to the analysis of trace Sudan black B. It could be detected at concentrations as low as 0.05 mg/L in standard solutions and 0.1 mg/kg in black rice extracts with the SERS method for measurement. The linear relationship between the intensity and concentration could be used for the quantitative detection of Sudan black B. The relation between dyeing time of black rice stained by Sudan black B solution and SERS intensity was studied which indirectly showed the effectiveness of the extraction method we designed. The results of the quantitative analysis reveal the practicability of using this method to detect Sudan black B in black rice. As a rapid and sensitive detection method, SERS can be extended to detect other food products and has a great application prospect in food safety inspection.

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.

Factors affecting the separation performance of proteins in capillary electrophoresis.

Capillary electrophoresis (CE) is an effective tool for protein separation and analysis. Compared with capillary gel electrophoresis (CGE), non-gel sieving capillary electrophoresis (NGSCE) processes the superiority on operation, repeatability and automaticity. Herein, we investigated the effect of polymer molecular weight and concentration, electric field strength, and the effective length of the capillary on the separation performance of proteins, and find that (1) polymer with high molecular weight and concentration favors the separation of proteins, although concentrated polymer hinders its injection into the channel of the capillary due to its high viscosity. (2) The resolution between the adjacent proteins decreases with the increase of electric field strength. (3) When the effective length of the capillary is long, the separation performance improves at the cost of separation time. (4) 1.4% (w/v) hydroxyethyl cellulose (HEC), 100 V/cm voltage and 12 cm effective length offers the best separation for the proteins with molecular weight from 14,400 Da to 97,400 Da. Finally, we employed the optimal electrophoretic conditions to resolve Lysozyme, Ovalbumin, BSA and their mixtures, and found that they were baseline resolved within 15 min.