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.

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 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.

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.

Although DNAs with single-nucleotide difference (e.g., 1000 and 1001 nt) can be resolved by capillary electrophoresis (CE), separation of the same length DNA with single-nucleotide difference is still a great challenge, which is caused by the structural or thermodynamical fluctuation of the sieving matrix. Here, a homogeneous tetra-arm poly (ethylene glycol) (Tetra-PEG) polymer network is synthesized in a capillary, and single monoprotic ssDNA is successfully separated in the capillary filled with cross-linked Tetra-PEG polymer. Results show that the separation performance in both slab-gel electrophoresis and CE is dramatically improved by Tetra-PEG gel than polyacrylamide gel as sieving matrix. Moreover, separation based on Tetra-PEG gel depends on not only the DNA size but also the chemical modification and DNA sequence, indicating that it is an ideal alternative to polyacrylamide gel.

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.

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.