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.

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.

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.

Polymerase chain reaction (PCR) is widely regarded as the gold standard for nucleic acid analysis; however, conventional thermal cycling limits its applicability in rapid and compact analytical systems. Here, we report an oscillating-flow microfluidic PCR method that enables rapid and flexible amplification by repeatedly shuttling the reaction mixture between two fixed-temperature zones. Unlike continuous-flow PCR, the proposed approach decouples PCR cycle number from microchannel geometry, allowing programmable cycling while reducing chip footprint. To enhance analytical reliability, polymer-assisted surface passivation using polyvinylpyrrolidone was employed to suppress nonspecific adsorption in polydimethylsiloxane (PDMS) microchannels, significantly improving amplification efficiency. Using Porphyromonas gingivalis and Treponema denticola as representative periodontal pathogens, 35-cycle amplification was completed within 20 min with reliable product yield. The proposed method advances oscillating-flow PCR toward a robust analytical strategy for rapid pathogen detection and related microfluidic nucleic acid analysis.