Probe-lengthening amplification (PLA) is a new nucleic-acid-amplification strategy that achieves decent selectivity by effectively avoiding nonspecific amplification. PLA has certain advantages such as a simple process of detection and an exponential accumulation of DNA copies. In addition, PLA has been employed for the detection of low-concentration DNA with a single-base differentiation ability [43]. Liu et al. described a PLA method involving an electrochemical biosensing strategy for ultrasensitive detection of genomic DNA in human serum [43]. The electrochemical signal was based on short probes for labelling with thiols and biotin; moreover, the operations were complex. Therefore, a new strategy was urgently required for the detection of nucleic acids based on PLA. A new strategy involving a combination of PLA and MCE was recently proposed [44]. This strategy exhibited sensitive detection of low-abundance genomic DNA; additionally, PLA was found to effectively avoid non-specific amplification. The target bacterial 16S rRNA gene was selected for this experiment. Meanwhile, auto-continuous MCE provided an ultra-high-throughput method for bacterial screening. Wang et al. proposed a strategy by combining PLA with MCE for the detection of five bacteria based on the analysis of 16S rRNA genes [44]. In this assay, four specific short probes were designed for a target bacterium by recognising the bacterial 16S rRNA gene. Fig. 5 shows a schematic of this method. The 16S rRNA genes of Enterococcus faecalis, E. coli, Staphylococcus aureus (S. aureus), Pseudomonas aeruginosa (P. aeruginosa), and Proteus mirabilis (P. mirabilis) were investigated using this approach. The results showed that sensitive detection of bacterial genes was realised based on the combination of PLA and MCE with a LOD of 30 fmol/L. This approach for the bacterial 16S rRNA gene has significant potential for application in the diagnosis of infection.

Among the recently developed nucleic acid assays for the detection of pathogenic bacteria, the polymerase chain reaction (PCR) has shown tremendous promise for the effective, simultaneous, quantitative, and rapid detection of pathogenic bacteria [45-48]. Although traditional methods of bacterial culture are simple and inexpensive, they are time-consuming [4,49]. Compared with traditional detection strategies, PCR-based techniques in combination with gel electrophoresis (GE) are being explored as alternative strategies for the detection of bacteria to reduce the detection time. However, gel electrophoresis exhibits low detection sensitivity and is tedious in terms of operation [50]. Therefore, combinations of PCR-based techniques and MCE are preferable for the detection of pathogenic bacteria, compared with those with GE, because of the obvious advantages of MCE such as rapid operation, easier miniaturisation, and higher accuracy in quantitative analysis of DNA fragments, as well as higher sensitivity and resolution [42,51,52]. Zhang et al. reported on the detection of three foodborne pathogenic bacteria (E. coli, S. aureus and S. Typhimurium) by MCE based on the PCR strategy. Primer pairs targeting these foodborne pathogenic bacteria were selected from the literature or using biosoft. The separation of these foodborne pathogenic bacteria PCR products was realised within 135 s with limits of 1.6–3.5 ng/µL. The LODs were as low as 62, 45 and 42 CFU/mL for S. aureus, E. coli and S. typhimurium, respectively [42]. Zhang et al. also reported a PCR-based strategy combined with MCE for the detection of three foodborne pathogenic bacteria (L. monocytogenes, E. coli, and S. typhimurium). The obtained LODs were as low as 68, 82 and 85 CFU/mL for L. monocytogenes, E. coli and S. typhimurium, respectively [53]. Zhu et al. employed PCR technology in combination with MCE to detect S. aureus, Proteus mirabilis and Enterobacter sakazakii (E. sakazakii) (Fig. 6) [54]. Li et al. developed a strategy for the rapid and specific detection of L. monocytogenes and Y. enterocolitica in food by combining MCE-laser-induced fluorescence (LIF) with duplex PCR [55]. Chen et al. described an approach involving MCE coupled with a simple-to-use space domain internal standard (SDIS) method for the rapid analysis of S. aureus [56]. Lee et al. investigated a novel paper-based capillary electrophoresis (pCE) microdevice that was fabricated by a mineral paper company. The microdevice was found to offer benefits in terms of being durable, waterproof, tear-resistant, simple to fabricate, lightweight, inexpensive, and disposable. The PCR products of E. coli O157:H7 and S. aureus were detected using the pCE microdevice. The LOD indicated that S. aureus and E. coli O157:H7 could be successfully analysed with 1.6 × 10² copies (0.5 pg) and 1.6 × 10² copies (0.5 pg) of the genomic DNA templates, respectively [57]. This highly sensitive MCE detection method requires smaller amounts of reagents and has a shorter analysis time. Therefore, PCR-based strategies combined with MCE can be employed for routine microbial detection in foods owing to these advantages.

PCR is considered the most powerful strategy for the detection of bacteria compared to traditional culture methods. However, a special PCR instrument had to be designed for amplification of target DNA or RNA in traditional PCR. Therefore, a new strategy was developed for performing PCR on a microchip. The advantages of performing PCR on a microchip, compared to traditional PCR, include high heat-transfer efficiency, excellent surface properties, cost-effective product design, and low reagent consumption. Rizzo et al. developed a rapid procedure for the molecular detection of P. aeruginosa, E. coli, S. epidermidis, and S. aureus by integrating pathogen capture in blood samples via peptides displayed in engineered M13 phage-coated paramagnetic beads with direct RT-PCR into microchips (Fig. 7) [58]. Tong et al. designed a fully portable and ultra-fast microchip-based PCR instrument as a rapid pathogen detection system. three functional components were incorporated into the designed instrument: electrical modules, heating, and an optical system. Two different real DNA samples, E. coli O157:H7 and Salmonella, were evaluated using this portable PCR machine with a DNA detection sensitivity as low as 1 pg/µL [13]. Ohlsson et al. designed an integrated microsystem for the first online acoustophoretic plasma generation from blood with seed particle-enabled trapping enrichment and PCR to rapidly detect bacteria in blood samples. The entire process, including rapid separation, enrichment, and detection of bacteria from blood, was completed in less than 2 h The LOD of Pseudomonas putida (P. putida) was found to be as low as 1000 bacteria/mL in whole blood [59]. Gorgannezhad et al. fabricated a glass-polydimethylsiloxane (PDMS)-glass sandwiched microchip device as a microfluidic PCR array for the real-time PCR-based detection of E. coli and Bacteroidales. Modified Triton X-100 was employed on the surface of PDMS to prevent undesirable protein adsorption and enhance PCR compatibility in the chip. This strategy has been successfully applied to microbial faecal source tracking in water [60].

Molecular diagnostics play an important role in medical diagnosis, biological studies, food safety, and environmental monitoring. However, the effective detection of low amounts of target nucleic acids in complex biological samples required signal amplification techniques; loop-mediated isothermal amplification (LAMP) [61,62], CHA [39,63], RCA [64,65], and strand displacement amplification (SDA) [66,67], have been developed in this regard. SDA is an isothermal amplification method that generally depends on an ssDNA template containing complementary bases to the endonuclease recognition sequence; however, the SDA method falls short in terms of detecting short nucleic acids. Therefore, a new strategy involving the use of a defective T-junction structure was employed to support the SDA (dT-SDA). T junctions, which were used as a type of stable architecture, were formed by three strands of DNA based on the Watson-Crick complementary base pairing principle [68,69]. Yuan et al. developed an ultrasensitive and specific detection method for nucleic acids using a defective T junction combined with an electrochemical sensing strategy. The results indicated that the high sensitivity of target DNA for electrochemical DNA biosensing was as low as 0.4 fmol/L [70]. Wang et al. exploited this benefit of the dT-SDA strategy to develop a dT-SDA approach combined with MCE for the detection of longer bacterial 16S rDNA. A schematic of this process is shown in Fig. 8. With respect to dT-SDA, a primer and an auxiliary probe were designed to identify the target gene based on the formation of a stable defective T-junction structure via proximity hybridisation. Numerous ssDNA products were produced by triggering cascade SDA cycling based on the defective T junctions. The ssDNA products obtained were separated and detected using MCE. The concentration of the target gene was found to be a function of the quantity of ssDNA product. The proposed dT-SDA strategy took only up to 2.5 h to complete, and exhibited satisfactory sensitivity with a minimum LOD of 10 pmol/L; therefore, this method is a potential strategy for effective bacterial screening and infection diagnosis [71].

Among the recently developed nucleic acid assays for the detection of pathogenic bacteria, the isothermal strand-displacement polymerase reaction (ISDPR) technique has been explored as a promising strategy for effective, simultaneous, quantitative, and rapid detection of pathogenic bacteria [72,73]. ISDPR is an enzyme-assisted amplification process in which ssDNA is replaced by the substrate-ssDNA duplex through the extension of an initiator strand; moreover, this extension is completely complementary to the toehold region of the substrate strand. Nucleic acid amplification is achieved by this process based on the generation of several substrate-initiator duplexes. Sequence modelling is straightforward and temperature regulation is not strictly required in the process of ISDPR. In addition, ISDPR can realise exponential amplification with prominent signal amplification in less than an hour [74,75]. Based on these advantages, various assays based on molecular beacons [76], electrochemistry [77], and fluorescence [78] have been used to detect bacteria via ISDPR amplification. Wang et al. first integrated ISDPR with MCE for the detection of the characteristic mecA gene in methicillin-resistant S. aureus (MRSA) [79]. Fig. 9 shows a schematic of the ISDPR signal recycling amplification in which the target DNA first binds to the hairpin probe (HP), which results in the DNA hairpin structure being opened. The target DNA is released by the primer hybridised with the probe in the presence of the Klenow fragment, exo-polymerase. Meanwhile, the released target DNA could open the next HP. Target recycling and amplification were thereby achieved. The amplified products of the HP-cDNA duplex were detected by MCE with a LOD of 12.3 pmol/L for the target DNA. The proposed strategy was successfully applied to detect methicillin-resistant S. aureus and could prove to be useful for detecting other bacteria owing to its rapid detection, ultrasensitivity, label-free ability, and straightforward operation.

Sensitive, rapid, and cost-effective detection of pathogenic bacteria is urgently needed in medical diagnosis, biological studies, food safety, and environmental monitoring. Therefore, loop-mediated isothermal amplification (LAMP) has been employed for the rapid detection of pathogenic bacteria to accommodate the increasing demand for rapid detection techniques [80,81]. Compared with the PCR technology, LAMP is an isothermal nucleic-acid amplification method that does not require a thermal cycler because of the use of a single temperature throughout the reaction. In addition, LAMP is more tolerant to crude samples than PCR. Moreover, LAMP is more suitable than PCR because the PCR strategy requires well-equipped laboratories, sophisticated instruments, and specially trained technicians, which may not be feasible in resource-limited settings. LAMP has been used with four to six primers and a strand-displacing DNA polymerase to perform cyclic amplification at a constant temperature throughout the reaction. LAMP has also been proven to be more sensitive, specific, stable, and faster than PCR [82,83]. LAMP assays integrated with microchips have been recently developed for rapid pathogen detection. Zhou et al. fabricated a dual-sample microfluidic chip integrated with the LAMP assay to simultaneously detect ten pathogenic microorganisms. This on-chip LAMP provided highly sensitive results with LODs ranging from 10^-1 pg/µL to 100 pg/µL for genomic DNA of tested bacteria, with run times of less than 30 min [80]. Wang et al. developed a versatile microfluidic chip platform integrated with LAMP for the simultaneous detection of S. pneumoniae and M. pneumonia. The platform used in this study was composed of a portable device and a polymer/paper microfluidic chip (Fig. 10). Magnetic particles were employed in the microfluidic chip to extract nucleic acids of pathogen genomic DNA. The DNA extraction was realised in ~15 min using this microfluidic chip. The genomic DNA of the pathogens was subjected to LAMP. A portable device was used to heat the microfluidic chip and record the results. The analytical sensitivity of the assay was as low as 2 fg for the target DNA [84]. Fu et al. developed a rapid on-chip gene quantification strategy based on LAMP-assisted PCR. The LAMP assays were employed to detect the target genes of E. coli and Enterococcus spp. using the most-probable-number approach. The colourimetric LAMP assay achieved a high sensitivity of as few as 4 copies/well and clear positive/negative observations on polymethyl methacrylate microchips [81]. Zhong et al. developed a microfluidic-based real-time fluorogenic LAMP system for the reliable and simple detection of Vibrio splendidus (V. splendidus). A LAMP primer set with six primers (arsB1) specifically targeting the arsB gene of V. splendidus was first designed in this study to test the portable microfluidic system. A linear standard curve for V. splendidus in a template concentration range of 5 × 10³ to 5 × 10⁶ CFU/mL was constructed [85]. Dou et al. fabricated a polymer/paper hybrid microfluidic biochip integrated with LAMP for the detection of three major types of meningitis-causing bacteria, S. pneumoniae, Neisseria meningitidis, and Haemophilus influenzae type b (Hib), with high sensitivity and specificity. A smartphone camera under a portable UV light source was used to observe the results, which could also be observed by the naked eye. In addition, this strategy achieved high sensitivity with LODs of a few DNA copies per LAMP zone for the three investigated meningitis-causing bacteria within 1 h, without using any specialised laboratory instrument. Therefore, the microfluidic approach has significant potential for the detection of pathogens [86]. Seo et al. developed a centrifugal microfluidic device to identify foodborne pathogen bacteria via a combination of LAMP and colourimetric detection using Eriochrome Black T (EBT). The centrifugal microfluidic system included five identical structures to realise the genetic analysis of 25 pathogen samples in a high-throughput manner. The primer mixtures and DNA sample solutions were mixed in the microchannels, followed by being subjected to LAMP. Three types of pathogenic bacteria (Salmonella typhimurium, Escherichia coli O157:H7, and Vibrio parahaemolyticus) were detected using the EBT-mediated colourimetric strategy. However, the results demonstrated that the genomic DNA of E. coli O157:H7 was specifically identified using primer sets in the LAMP reaction based on identification with the naked eye, with the entire process lasting 60 min. The developed microsystem enabled adequate testing of foodborne pathogens with high simplicity and high speed without using any bulky and expensive instrumentation [61].

S. aureus is a Gram-positive rod-shaped bacterium found in air, dust, soil, and water that can cause various diseases in humans [87,88]. Rapid and accurate bacterial detection strategies that can effectively monitor the quality of food supplies are urgently required to enable effective control of microbial contamination of food and ensure food safety. Conventional microbial culture methods used for the identification and quantification of pathogens are time-consuming and extremely labour-intensive. Microchips are a powerful strategy to solve this issue because of their high information exportation, rapid response, automatic sampling, and high-throughput [89]. Zhang et al. developed a strategy that utilised Cy5 to label vancomycin (Cy5-Van) for the specific binding and detection of S. aureus, as shown in Fig. 11. The LOD of S. aureus obtained using this strategy was as low as 981 CFU/mL [89]. Song et al. developed a microfluidic platform for the detection of S. aureus based on fluorescence labelling. This microfluidic platform involved a microbial culture and S. aureus antibodies coated on the diameter of 50–90 µm microspheres to study the effect of capturing S. aureus. This platform quantitatively monitored bacteria with a LOD of 1.5 × 10¹ CFU/µL. The results indicated that the microfluidic chip used in this study was sensitive, accurate, portable, and could be further employed for investigating the detection of bacteria [90].

The rapid and sensitive detection of E. coli in water and food samples is extremely important for food safety. Several methods based on PCR [91] and electrochemical technologies [92] have been used for the detection of E. coli. One of these methods for the detection of E. coli involves measuring the activity of β-galactosidase (β-gal), which is frequently used as a marker for coliforms [93,94]. To date, β-gal has been used to examine the concentration of E. coli in food and water samples. Electrochemical methods [95], colourimetry [96], and fluorescent substrates [97] have been applied for the detection of E. coli based on the activity of β-gal. Wang et al. developed a sensitive strategy based on the activity of β-gal in which the measurement of 4-aminophenol was realised by MCE, which was used further to indicate E. coli. 4-Aminophenol is obtained via the hydrolysis of the 4-aminophenyl-β-d-galactopyranoside (PAPG) substrate by β-gal [98]. A schematic of the activity of β-gal activity is shown in Fig. 12. The concentration of E. coli was found to be proportional to the quantity of 4-aminophenol. This microfluidic platform used the field-amplified sample injection (FASI) method to increase the sensitivity of 4-aminophenol. The results indicated that the LOD reached 10 CFU/mL based on the fluorescence responses of 4-aminophenol, and this strategy achieved the detection of E. coli at a level as low as 23 CFU/mL in river water. Therefore, this strategy provided new insights into the detection of bacteria.

MCE has become an attractive strategy for the analysis of pathogenic bacteria because of its ease of operation, high throughput, cost-effectiveness, and high sensitivity, compared with those of traditional culture or molecular methods such as PCR. However, currently reported MCE devices are facing significant challenges for the rapid detection of pathogenic bacteria. Therefore, various new microfluidic devices have been developed and applied for the detection of pathogens. Nguyen et al. developed a smartphone-based electric power system for microcapillary electrophoresis (µCE) on a chip (Fig. 13), which exhibits advantages such as improved portability of the µCE device and simple electrical operations. This system achieved the detection of 4.03 ng/µL of E. coli and 3.8 ng/µL of S. typhimurium [99]. Altintas et al. developed a fully automated microfluidic-based electrochemical biosensor for the quantification of E. coli in water samples (Fig. 14). This strategy was based on a nanomaterial-modified assay with high sensitivity and specificity, and exhibited tremendous potential for pathogen detection [100]. Fang et al. fabricated an integrated microfluidic chip (IMC) that included a membrane-based filtration module, a bacteria-capturing module, and a miniature PCR module for bacterial identification. This microdevice was applied for the rapid isolation of both Gram-negative and Gram-positive bacteria from blood. All white-blood cells and 99.5% of red blood cells were separated from the bacteria using a filtration module. The LOD for the PCR assay was 5 CFU per reaction, and the entire process was complete within 4 h [101]. Chen et al. developed a finger-actuated microfluidic chip (µFAchip) integrated with gel-based loop-mediated isothermal amplification (gLAMP) for the simultaneous detection of multiple pathogens. The designed µFAchip comprised three PDMS layers that could be used for finger-actuated operation to mix the sample and reagents in a controlled manner. An on-chip LAMP reaction was conducted on a miniaturised heater for realising point-of-care testing (POCT), and the resulting imaging was recorded using a portable fluorescence imaging system (Fig. 15). The developed µFAchip achieved a detection of as low as 1.6 cells with high selectivity and sensitivity, and provided a rapid and easy-to-operate platform for POCT-related applications of multiplexed pathogens [102]. Yamaguchi et al. developed a microfluidic device comprising on-chip fluorescent staining and semi-automated counting of target microbial cells based on fluorescent antibody staining. In addition, the microfluidic device was integrated with a self-made portable system for rapid and accurate counting of bacterial cells in freshwater [103]. Jin et al. developed a self-priming compartmentalisation (SPC) microdevice that was integrated into the LAMP system for enabling high-throughput visual detection. The microfluidic chip comprised a self-filling microwell array. The reaction solution was introduced from the access hole in the chip. In addition, the method employed for avoiding cross-contamination and ensuring the accuracy of the results was found to depend on the independent and closed wells. This approach achieved the simultaneous determination of six foodborne pathogens within 1 h [104]. Li et al. developed a strategy in which rapid bacterial identification in urine samples was achieved based on a microchannel silicon-nanowire microfluidic chip coupled with matrix assisted laser desorption ionisation-time of flight mass spectrometry (MALDI-TOF MS). Bacteria were detected at concentrations as low as 10³ CFU/mL based on culturing urine samples for 4 h [105]. Khan et al. developed an electrically-receptive thermally-responsive (ER-TR) sensor chip that was composed of simple filter paper used as a substrate based on poly(N-isopropylacrylamide)-graphene nanoplatelets. Au electrodes were evaporated to realise real-time capture of bacteria such as S. mutans, B. subtilis and E. coli [106]. Oh et al. developed a centrifugal microfluidic device for fully automated and colourimetric foodborne pathogen detection. DNA extraction and purification, DNA amplification and amplicon detection were performed on a single disc. Silica microbeads were used for the extraction and purification of bacterial genomic DNA from bacteria-contaminated milk samples. Four types of foodborne pathogens (S. typhimurium, E. coli O157:H7, L. monocytogenes and V. parahaemolyticus) were selected and subjected to LAMP to amplify specific genes of these bacteria. The results of the LAMP reactions relied on colourimetric detection mediated by a metal indicator. The results obtained indicated its potential for detecting foodborne pathogens from a contaminated milk sample, with a LOD of 10 bacterial cells achieved in 65 min [107]. Yang et al. developed a non-biofouling polyethylene glycol (PEG)-based microfluidic chip integrated with a functionalised nanoporous alumina membrane. The anti-E. coli O157:H7 antibody and anti-S. aureus antibody were individually immobilised on two nanoporous alumina membranes. E. coli O157:H7 and Staphylococcus aureus were specific targets in terms of bacterial detection, with a LOD of approximately 10² CFU/mL [108]. Shen et al. developed a herringbone microfluidic chip integrated with vancomycin-modified magnetic beads (herringbone-VMB microchip) to enrich pathogens, which were identified by MALDI-TOF MS [16]. Wang et al. achieved online and sensitive detection of Salmonella using a microfluidic biosensor that included immunomagnetic separation, fluorescence labelling, and smartphone video processing (Fig. 16). The LOD indicated that Salmonella could be successfully analysed at 58 CFU/mL using this proposed biosensor [109]. Li et al. developed a fully hand-powered centrifugal miniaturised nucleic-acid amplification testing platform, which comprised sample preparation, strand exchange amplification (SEA), and visual fluorescence detection as components. Sample preparation, reagent manipulation, mixing, SEA reaction, and fluorescence signal observations were realised in a single chip for the first time in this study. A schematic of the hand-powered microfluidic centrifugal platform is shown in Fig. 17. The microfluidic platform achieved a DNA or RNA LOD as low as 10 CFU/g via enrichment within 1 h [110]. Kwon et al. developed a modified herringbone microfluidic chip prepared using gold-nanoparticle-embedded PDMS. This microfluidic device was found to be applicable for adsorbing and destroying airborne bacteria and enriching and extracting DNA from pathogenic bacteria (S. aureus, E. coli, P. aeruginosa and Bacillus cereus) in air samples [111]. Kim et al. developed a micromachined microfluidic device that comprised cell focusing, dielectrophoretic sensing, and impedance measurement for label-free detection of E. coli in drinking water. The proposed system was found to achieve high throughput and exhibit a low LOD with enumerating populations as low as 300 CFU/mL [112].