In the decade after being awarded the Nobel Prize in Chemistry in 2002, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been widely used as an analytical chemistry tool for the detection of large and small molecules (e.g., polymers, proteins, peptides, nucleic acids, amino acids, lipids, etc.) and for clinical analysis and research (e.g., pathogen identification, genetic disorders screening, cancer diagnosis, etc.). In view of the fast development of MALDI-TOF MS in clinical usage, this review systematically summarizes the most important applications of MALDI-TOF MS in clinical analysis and research by analyzing MALDI TOF MS-related reviews collected in the Web of Science database. On the basis of the analysis of keyword co-occurrence of over 2000 review articles, four themes consisting of “pathogen identification”, “disease diagnosis”, “nucleic acids analysis”, and “small molecules analysis” were found. For each theme, the review further outlined their application implications, analytical methods, and systems as well as limitations that need to be addressed. Overall, the review summarizes and elaborates on the clinical applications of MALDI-TOF MS, providing a comprehensive picture for researchers embarking on MALDI TOF MS-related clinical analysis and research.

MALDI-TOF Mass Spectrometry in Clinical Analysis and Research

Rapid and accurate detection of foodborne pathogens is a powerful and effective strategy to prevent the outbreak of foodborne diseases. Conventional detection methods like the plate counting and molecular diagnosis strategies are still not suitable for timely and rapid on-site detection due to the cumbersome operations, requirement of trained operators and sophisticated instruments. Microfluidics provide a powerful tool for pathogen detection due to the intrinsic advantages including easy integration, miniaturization, portability, and small sample consumption. Up to now, many research works have reported the combination of microfluidics with various detection techniques and signal amplification technologies to achieve the rapid and sensitive detection of foodborne pathogens. In this review, we mainly summarized the significant developed microfluidic based devices for pathogenic bacteria detection over the past five years, including (1) integration of sample preparation techniques like cell capture and enrichment, and nucleic acid related sample preparation methods on chip; (2) various detection methods, such as colorimetric, fluorescence, chemiluminescence, and mass spectrometry; (3) microfluidic paper-based analytical devices, particularly for the applications in resource-limited settings, remote areas or on-site detection. The limitations and advantages of these methods were summarized and discussed in order to comprehensively understand the latest microfluidic-based detection techniques for foodborne pathogens. Finally, the future challenges and directions for pathogens detection based on microfluidic based biosensors were also discussed and summarized. 

Recent advances in microfluidic devices for foodborne pathogens detection

Sensitive and rapid detection of pathogenic bacteria is essential for effective source control and prevention of microbial infectious diseases. However, it remains a substantial challenge to rapidly detect bacteria at the single-cell level. Herein, we present an electrochemical sandwich sensor for highly selective and ultrasensitive detection of a single bacterial cell based on dual recognition by the bacteria-imprinted polymer film (BIF) and aptamer. The BIF was used as the capture probe, which was in situ fabricated on the electrode surface within 15 min via electropolymerization. The aptamer and electroactive 6-(Ferrocenyl)hexanethiol cofunctionalized gold nanoparticles (Au@Fc-Apt) were employed as the signal probe. Once the target bacteria were anchored on the BIF-modified electrode, the Au@Fc-Apt was further specifically bound to the bacteria, generating enhanced current signals for ultrasensitive detection of Staphylococcus aureus down to a single cell in phosphate buffer solution. Even in the complex milk samples, the sensor could detect as low as 10 CFU mL-1 of S. aureus without any complicated pretreatment except for 10-fold dilution. Moreover, the current response to the target bacteria was hardly affected by the coexisting multiple interfering bacteria, whose number is 30 times higher than the target, demonstrating the excellent selectivity of the sensor. Compared with most reported sandwich-type electrochemical sensors, this assay is more sensitive and more rapid, requiring less time (1.5 h) for the sensing interface construction. By virtue of its sensitivity, rapidity, selectivity, and cost-effectiveness, the sensor can serve as a universal detection platform for monitoring pathogenic bacteria in fields of food/public safety.

Dual Synthetic Receptor-Based Sandwich Electrochemical Sensor for Highly Selective and Ultrasensitive Detection of Pathogenic Bacteria at the Single-Cell Level.

The infection induced by harmful bacteria has become one of the leading public health concerns worldwide, threatening social health and the economy. In addition, new and multidrug-resistant pathogens are continuously emerging with the abuse of antibiotics. Therefore, there is an urgent need to develop advanced platforms for precise detection and antibiotic-free inhibition of pathogens toward large-scale clinical applications. Advanced materials with unique structure and surface chemistry have emerged in bacterial detection and inhibition, while still lacking explicit elaboration of the connections between material properties and analytical/inhibition performance. This review describes the recent advancements regarding the precise detection of pathogens based on advanced materials incorporated with analytical approaches, including colorimetry, fluorescence, and molecular fingerprinting platforms. The antibacterial applications of advanced material-assisted platforms are then reviewed from three mechanism concepts of physical, chemical, and synergetic damage. The review ends with ideas on current limitations and some potential perspectives, thus may serving as a handbook to guide future research. 

Advanced materials for precise detection and antibiotic-free inhibition of bacteria

Genital mycoplasmas can break the cervical barrier and cause intraamniotic infection and preterm birth. This study developed a six-chamber vagina-cervix-decidua-organ-on-a-chip (VCD-OOC) that recapitulates the female reproductive tract during pregnancy with culture chambers populated by vaginal epithelial cells, cervical epithelial and stromal cells, and decidual cells. Cells cultured in VCD-OOC were characterized by morphology and immunostaining for cell-specific markers. We transferred the media from the decidual cell chamber of the VCD-OOC to decidual cell chamber in feto-maternal interface organ-on-a-chip (FMi-OOC), which contains the fetal membrane layers. An ascending Ureaplasma parvum infection was created in VCD-OOC. U. parvum was monitored for 48 h post-infection with their cytotoxicity (LDH assay) and inflammatory effects (multiplex cytokine assay) in the cells tested. An ascending U. parvum infection model of PTB was developed using CD-1 mice. The cell morphology and expression of cell-specific markers in the VCD-OOC mimicked those seen in lower genital tract tissues. U. parvum reached the cervical epithelial cells and decidua within 48 h and did not cause cell death in VCD-OOC or FMi-OOC cells. U. parvum infection promoted minimal inflammation, while the combination of U. parvum and LPS promoted massive inflammation in the VCD-OOC and FMi-OOC cells. In the animal model, U. parvum vaginal inoculation of low-dose U. parvum did not result in PTB, and even a high dose had only some effects on PTB (20%). However, intra-amniotic injection of U. parvum resulted in 67% PTB. We report the colonization of U. parvum in various cell types; however, inconsistent, and low-grade inflammation across multiple cell types suggests poor immunogenicity induced by U. parvum. 

Modeling ascending Ureaplasma parvum infection through the female reproductive tract using vagina‐cervix‐decidua‐organ‐on‐a‐chip and feto‐maternal interface‐organ‐on‐a‐chip

Bacterial infections cause considerable morbidity and expensive healthcare costs. The prescription of broad-spectrum antimicrobial drugs results in failure of treatment or overtreatment and exacerbates the spread of multidrug-resistant pathogens. There is an emergent demand for rapid and accurate methods to identify pathogens and conduct personalized therapy. Here, we develop a herringbone microfluidic chip integrated with vancomycin modified magnetic beads (herringbone-VMB microchip) to enrich pathogens. The enriched pathogens are identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The herringbone-VMB microchip applies passive mixing of bacterial samples by generating microvortices, which significantly enhances the interaction between bacteria and vancomycin modified magnetic beads and leads to more efficient enrichment compared to in-tube extraction. Four common pathogens in urinary tract infections are utilized to validate the method, and the capture efficiency of the bacteria from urine is up to 90%. The whole procedure takes 1.5 hours from enrichment to identification. This method shows potential in shortening the turnaround time in the clinical diagnosis of bacterial infections.

Highly efficient enrichment and identification of pathogens using a herringbone microfluidic chip and by MALDI-TOF mass spectrometry.

Cervix chip mimicking cervical microenvironment for quantifying sperm locomotion.

Before fertilization, sperms adhere to oviductal epithelium cells, and only a restrictive number of winner sperms can escape to reach the egg. To study the sperm escape behavior from the oviductal surface, we developed a microfluidic chip to fabricate an adhesive surface and to create a gradient of progesterone (P4) for mimicking the oviduct microenvironment in vivo. We identified three sperm motion patterns in such a microenvironment─anchored spin, run-and-spin, and escaped mode. By using kinetic analysis, we verified the hypothesis that the responsive rotation energy anchored with the adhered sperm head determines whether the sperm is trapped or detaching, which is defined as the hammer flying strategy of successful escape after accumulating energy in the process of rotating. Intriguingly, this hammer-throw escaping is able to be triggered by the P4 biochemical stimulation. Our results revealed the tangled process of sperm escape before fertilization in the ingenious microfluidic system.

Escaping Behavior of Sperms on the Biomimetic Oviductal Surface.

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Highly efficient enrichment and identification of pathogens using a herringbone microfluidic chip and by MALDI-TOF mass spectrometry.

Cervix chip mimicking cervical microenvironment for quantifying sperm locomotion.

Escaping Behavior of Sperms on the Biomimetic Oviductal Surface.