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1. Isothermal Amplification and Lateral Flow Nucleic Acid Test for the Detection of Shiga Toxin-Producing Bacteria for Food Monitoring

   https://doi.org/10.3390/chemosensors10060210  

   Petrucci, Sabrina; Dikici, Emre; Daunert, Sylvia; Deo, Sapna K. (June 2022, Chemosensors)

   Foodborne bacteria have persisted as a significant threat to public health and to the food and agriculture industry. Due to the widespread impact of these pathogens, there has been a push for the development of strategies that can rapidly detect foodborne bacteria on-site. Shiga toxin-producing E. coli strains (such as E. coli O157:H7, E. coli O121, and E. coli O26) from contaminated food have been a major concern. They carry genes stx1 and/or stx2 that produce two toxins, Shiga toxin 1 and Shiga toxin 2, which are virulent proteins. In this work, we demonstrate the development of a rapid test based on an isothermal recombinase polymerase amplification reaction for two Shiga toxin genes in a single reaction. Results of the amplification reaction are visualized simultaneously for both Shiga toxins on a single lateral flow paper strip. This strategy targets the DNA encoding Shiga toxin 1 and 2, allowing for broad detection of any Shiga toxin-producing bacterial species. From sample to answer, this method can achieve results in approximately 35 min with a detection limit of 10 CFU/mL. This strategy is sensitive and selective, detecting only Shiga toxin-producing bacteria. There was no interference observed from non-pathogenic or pathogenic non-Shiga toxin-producing bacteria. A detection limit of 10 CFU/mL for Shiga toxin-producing E. coli was also obtained in a food matrix. This strategy is advantageous as it allows for timely identification of Shiga toxin-related contamination for quick initial food contamination assessments. 

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2. A microfluidic fully paper-based analytical device integrated with loop-mediated isothermal amplification and nano-biosensors for rapid, sensitive, and specific quantitative detection of infectious diseases

   https://doi.org/10.1039/D2LC00834C  

   Tavakoli, Hamed; Hirth, Elisabeth; Luo, Man; Sharma Timilsina, Sanjay; Dou, Maowei; Dominguez, Delfina C.; Li, XiuJun (November 2022, Lab on a Chip)

   Bacterial meningitis, an infection of the membranes (meninges) and cerebrospinal fluid (CSF) surrounding the brain and spinal cord, is one of the major causes of death and disability worldwide. Higher case-fatality rates and short survival times have been reported in developing countries. Hence, a quick, straightforward, and low-cost approach is in great demand for the diagnosis of meningitis. In this research, a microfluidic fully paper-based analytical device (μFPAD) integrated with loop-mediated isothermal amplification (LAMP) and ssDNA-functionalized graphene oxide (GO) nano-biosensors was developed for the first time for a simple, rapid, low-cost, and quantitative detection of the main meningitis-causing bacteria, Neisseria meningitidis ( N. meningitidis ). The results can be successfully read within 1 hour with the limit of detection (LOD) of 6 DNA copies per detection zone. This paper device also offers versatile functions by providing a qualitative diagnostic analysis ( i.e. , a yes or no answer), confirmatory testing, and quantitative analysis. These features make the presented μFPAD capable of a simple, highly sensitive, and specific diagnosis of N. meningitis . Furthermore, this microfluidic approach has great potential in the rapid detection of a wide variety of different other pathogens in low-resource settings. 

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3. Analytical methods for detection of Zika virus

   https://doi.org/10.1557/mrc.2017.20  

   Yang, Kai-Hung; Narayan, Roger J. (March 2017, MRS Communications)

   Due to the recent outbreak of the Zika virus (ZIKV) in several regions, rapid, and accurate methods to diagnose Zika infection are in demand, particularly in regions that are on the frontline of a ZIKV outbreak. In this paper, three diagnostic methods for ZIKV are considered. Viral isolation is the gold standard for detection; this approach can involve incubation of cell cultures. Serological identification is based on the interactions between viral antigens and immunoglobulin G or immunoglobulin M antibodies; cross-reactivity with other types of flaviviruses can cause reduced specificity with this approach. Molecular confirmation, such as reverse transcription polymerase chain reaction (RT–PCR), involves reverse transcription of RNA and amplification of DNA. Quantitative analysis based on real-time RT–PCR can be undertaken by comparing fluorescence measurements against previously developed standards. A recently developed programmable paper-based detection approach can provide low-cost and rapid analysis. These viral identification and viral genetic analysis approaches play crucial roles in understanding the transmission of ZIKV. 

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4. Porous silicon on paper sensors: factors affecting biosensing performance

   https://doi.org/10.1117/12.3048667  

   An, Huijin; Laibinis, Paul E; Locke, Andrea K; Weiss, Sharon M (March 2025, SPIE)

   Miller, Benjamin L; Weiss, Sharon M; Danielli, Amos (Ed.)

   A paper-based biosensor integrating a functionalized porous silicon (PSi) membrane as the active sensing element for quantifiable protein detection has been developed. For similar short-time exposures to an analyte, improved molecular transport in PSi membranes when on paper leads to larger signal changes compared to traditional PSi films that remain on a silicon substrate. In this work, we discuss controlling the incubation time of the analyte and the overall testing time of the sensor by incorporating different combinations of wicking and absorbent paper beneath the PSi membrane. With this control, the PSi-on-paper sensor platform has the potential to serve as an effective low-cost rapid diagnostic test with highly sensitive, quantitative readout for a wide range of analytes. 

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5. Label-free fluorescence assay coupled exonuclease reaction and SYBR Green I for the detection of T4 polynucleotide kinase activity

   https://doi.org/10.1039/c9ay02283j  

   Wu, Xu; He, Shuyi; Zhao, Julia Xiaojun (February 2020, Analytical Methods)

   A sensitive label-free fluorescence assay for monitoring T4 polynucleotide kinase (T4 PNK) activity and inhibition was developed based on a coupled λ exonuclease cleavage reaction and SYBR Green I. In this assay, a double-stranded DNA (dsDNA) was stained with SYBR Green I and used as a substrate for T4 PNK. After the 5′-hydroxyl termini of the dsDNA was phosphorylated by the T4 PNK, the coupled λ exonuclease began to digest the dsDNA to form mononucletides and single-stranded DNA (ssDNA). At this moment, the fluorescence intensity of the SYBR Green I decreased because of less affinity with ssDNA than dsDNA. The decreasing extent was proportional to the concentration of the T4 PNK. After optimization of the detection conditions, including the concentration of ATP, amount of λ exonuclease and reaction time, the activity of T4 PNK was monitored by the fluorescence measurement, with the limit of detection of 0.11 U mL −1 and good linear correlation between 0.25–1.00 U mL −1 ( R 2 = 0.9896). In this assay, no label was needed for fluorescence detection. Moreover, the inhibition behaviors of the T4 PNK's inhibitors were evaluated by this assay. The result indicated the potential of using this assay for monitoring of the phosphorylation-related process. 

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