Multiplex Detection of Foodborne Pathogens using 3D Nanostructure Swab and Deep Learning-Based Classification of Raman Spectra.

Proactive management of foodborne illness requires routine surveillance of foodborne pathogens, which requires developing simple, rapid, and sensitive detection methods. Here, a strategy is presented that enables the detection of multiple foodborne bacteria using a 3D nanostructure swab and deep learning-based Raman signal classification. The nanostructure swab efficiently captures foodborne pathogens, and the portable Raman instrument directly collects the Raman signals of captured bacteria. a deep learning algorithm has been demonstrated, 1D convolutional neural network with binary labeling, achieves superior performance in classifying individual bacterial species. This methodology has been extended to mixed bacterial populations, maintaining accuracy close to 100%. In addition, the gradient-weighted class activation mapping method is used to provide an investigation of the Raman bands for foodborne pathogens. For practical application, blind tests are conducted on contaminated kitchen utensils and foods. The proposed technique is validated by the successful detection of bacterial species from the contaminated surfaces. The use of a 3D nanostructure swab, portable Raman device, and deep learning-based classification provides a powerful tool for rapid identification (≈5 min) of foodborne bacterial species. The detection strategy shows significant potential for reliable food safety monitoring, making a meaningful contribution to public health and the food industry.

Kinetics of adaptive immune responses after administering mRNA-Based COVID-19 vaccination in individuals with and without prior SARS-CoV-2 infections.

OBJECTIVE: We aimed to compare the adaptive immune response in individuals with or without prior SARS-CoV-2 infections following the administration of mRNA-based COVID-19 vaccines. METHODS: A total of 54 participants with ages ranging from 37 to 56 years old, consisting of 23 individuals without a history of SARS-CoV-2 infection (uninfected group) and 31 individuals with prior infection of SARS-CoV-2 (infected group) who have received two doses of mRNA SARS-CoV-2 vaccines were enrolled in this study. We measured the IFN-γ level upon administration of BNT162b2 (PF) or mRNA-1273 (MO) by QuantiFERON SARS-CoV-2. The production of neutralizing antibodies was evaluated by a surrogate virus neutralization assay, and the neutralizing capacity was assessed by a plaque reduction neutralization test (PRNT50). The immune response was compared between the two groups. RESULTS: A significantly higher level of IFN-γ (p < 0.001) and neutralization antibodies (p < 0.001) were observed in the infected group than those in the uninfected group following the first administration of vaccines. The infected group demonstrated a significantly higher PRNT50 titer than the uninfected group against the Wuhan strain (p < 0.0001). Still, the two groups were not significantly different against Delta (p = 0.07) and Omicron (p = 0.14) variants. Following the second vaccine dose, T- and B-cell levels were not significantly increased in the infected group. CONCLUSION: A single dose of mRNA-based COVID-19 vaccines would boost immune responses in individuals who had previously contracted SARS-CoV-2.

SERS-ELISA using silica-encapsulated Au core-satellite nanotags for sensitive detection of SARS-CoV-2.

The sensitive detection of viruses is key to preventing the spread of infectious diseases. In this study, we develop a silica-encapsulated Au core-satellite (CS@SiO2) nanotag, which produces a strong and reproducible surface-enhanced Raman scattering (SERS) signal. The combination of SERS from the CS@SiO2 nanotags with enzyme-linked immunosorbent assay (ELISA) achieves a highly sensitive detection of SARS-CoV-2. The CS@SiO2 nanotag is constructed by assembling 32 nm Au nanoparticles (AuNPs) on a 75 nm AuNP. Then the core-satellite particles are encapsulated with SiO2 for facile surface modification and stability. The SERS-ELISA technique using the CS@SiO2 nanotags provides a great sensitivity, yielding a detection limit of 8.81 PFU mL-1, which is 10 times better than conventional ELISA and 100 times better than lateral flow assay strip method. SERS-ELISA is applied to 30 SARS-CoV-2 clinical samples and achieved 100% and 55% sensitivities for 15 and 9 positive samples with cycle thresholds < 30 and > 30, respectively. This new CS@SiO2-SERS-ELISA method is an innovative technique that can significantly reduce the false-negative diagnostic rate for SARS-CoV-2 and thereby contribute to overcoming the current pandemic crisis.

Ultrasensitive Isothermal Detection of SARS-CoV-2 Based on Self-Priming Hairpin-Utilized Amplification of the G-Rich Sequence.

The outbreak of the novel coronavirus disease 2019 (COVID-19) pandemic induced by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused millions of fatalities all over the world. Unquestionably, the effective and timely testing for infected individuals is the most imperative for the prevention of the ongoing pandemic. Herein, a new method was established for detecting SARS-CoV-2 based on the self-priming hairpin-utilized isothermal amplification of the G-rich sequence (SHIAG). In this strategy, the target RNA binding to the hairpin probe (HP) was uniquely devised to lead to the self-priming-mediated extension followed by the continuously repeated nicking and extension reactions, consequently generating abundant G-rich sequences from the intended reaction capable of producing fluorescence signals upon specifically interacting with thioflavin T (ThT). Based on the unique isothermal design concept, we successfully identified SARS-CoV-2 genomic RNA (gRNA) as low as 0.19 fM with excellent selectivity by applying only a single HP and further verified its practical diagnostic capability by reliably testing a total of 100 clinical specimens for COVID-19 with 100% clinical sensitivity and specificity. This study would provide notable insights into the design and evolution of new isothermal strategies for the sensitive and facile detection of SARS-CoV-2 under resource constraints.

Electrospun Nanofibrous Membrane-Based Colorimetric Device for Rapid and Simple Screening of Amphetamine-Type Stimulants in Drinks.

With the growth of drug-facilitated crimes, prevention has become increasingly important. Although various drug detection technologies exist, most focus on postconsumption detection. However, the prevention of drug-facilitated crimes requires technology for the quick and easy detection of amphetamine-type stimulants (ATSs) before ingestion. Herein, drug screening kits (DSKs) were developed for the simple detection of ATSs in drinks. The DSKs consisted of polydiacetylene nanofiber-based paper sensors fabricated by electrospinning with 10,12-pentacosadiynoic acid (PCDA) and PCDA-dopamine as sensing materials that can bind ATSs via hydrogen bonding and π-π interactions. Dropping a drink on the DSK provided an immediate visual indication of the presence of ATSs. When ATSs were present in the drink, the color of the DSK clearly changed from blue to red, with the increase in red intensity being more than twofold greater than that observed when water alone was tested. Notably, the result could be confirmed by the naked eye without any analytical instrumentation. A color change indicating the presence of ATSs was successfully observed in various alcoholic and nonalcoholic drinks. These results indicate the potential of DSKs for preventing drug-facilitated crimes caused by unwanted drug intake.

SERS-based dual-mode DNA aptasensors for rapid classification of SARS-CoV-2 and influenza A/H1N1 infection.

We developed a dual-mode surface-enhanced Raman scattering (SERS)-based aptasensor that can accurately diagnose and distinguish severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza A/H1N1 at the same time. Herein, DNA aptamers that selectively bind to SARS-CoV-2 and influenza A/H1N1 were immobilized together on Au nanopopcorn substrate. Raman reporters (Cy3 and RRX), attached to the terminal of DNA aptamers, could generate strong SERS signals in the nanogap of the Au nanopopcorn substrate. Additionally, the internal standard Raman reporter (4-MBA) was immobilized on the Au nanopopcorn substrate along with aptamer DNAs to reduce errors caused by changes in the measurement environment. When SARS-CoV-2 or influenza A virus approaches the Au nanopopcorn substrate, the corresponding DNA aptamer selectively detaches from the substrate due to the significant binding affinity between the corresponding DNA aptamer and the virus. As a result, the related SERS intensity decreases with increasing target virus concentration. Thus, it is possible to determine whether a suspected patient is infected with SARS-CoV-2 or influenza A using this SERS-based DNA aptasensor. Furthermore, this sensor enables a quantitative evaluation of the target virus concentration with high sensitivity without being affected by cross-reactivity. Therefore, this SERS-based diagnostic platform is considered a conceptually new diagnostic tool that rapidly discriminates against these two respiratory diseases to prevent their spread.

Sensitive and reproducible detection of SARS-CoV-2 using SERS-based microdroplet sensor.

Surface-enhanced Raman scattering (SERS)-based assays have been recently developed to overcome the low detection sensitivity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). SERS-based assays using magnetic beads in microtubes slightly improved the limit of detection (LoD) for SARS-CoV-2. However, the sensitivity and reproducibility of the method are still insufficient for reliable SARS-CoV-2 detection. In this study, we developed a SERS-based microdroplet sensor to dramatically improve the LoD and reproducibility of SARS-CoV-2 detection. Raman signals were measured for SERS nanotags in 140 droplets passing through a laser focal volume fixed at the center of the channel for 15 s. A comparison of the Raman signals of SERS nanotags measured in a microtube with those measured for multiple droplets in the microfluidic channel revealed that the LoD and coefficient of variation significantly improved from 36 to 0.22 PFU/mL and 21.2% to 1.79%, respectively. This improvement resulted from the ensemble average effects because the signals were measured for SERS nanotags in multiple droplets. Moreover, the total assay time decreased from 30 to 10 min. A clinical test was performed on patient samples to evaluate the clinical efficacy of the SERS-based microdroplet sensor. The assay results agreed well with those measured by the reverse transcription-polymerase chain reaction (RT-PCR) method. The proposed SERS-based microdroplet sensor is expected to be used as a new point-of-care diagnostic platform for quick and accurate detection of SARS-CoV-2 in the field.

FRET-based hACE2 receptor mimic peptide conjugated nanoprobe for simple detection of SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has led to a pandemic of acute respiratory disease, namely coronavirus disease (COVID-19). This disease threatens human health and public safety. Early diagnosis, isolation, and prevention are important to suppress the outbreak of COVID 19 given the lack of specific antiviral drugs to treat this disease and the emergence of various variants of the virus that cause breakthrough infections even after vaccine administration. Simple and prompt testing is paramount to preventing further spread of the virus. However, current testing methods, namely RT-PCR, is time-consuming. Binding of the SARS-CoV-2 spike (S) glycoprotein to human angiotensin-converting enzyme 2 (hACE2) receptor plays a pivotal role in host cell entry. In the present study, we developed a hACE2 mimic peptide beacon (COVID19-PEB) for simple detection of SARS-CoV-2 using a fluorescence resonance energy transfer system. COVID19-PEB exhibits minimal fluorescence in its ''closed'' hairpin structure; however, in the presence of SARS-CoV-2, the specific recognition of the S protein receptor-binding domain by COVID19-PEB causes the beacon to assume an ''open'' structure that emits strong fluorescence. COVID19-PEB can detect SARS-CoV-2 within 3 h or even 50 min and exhibits strong fluorescence even at low viral concentrations, with a detection limit of 4 × 103 plaque-forming unit/test. Furthermore, in SARS-CoV-2-infected patient samples confirmed using polymerase chain reaction, COVID19-PEB accurately detected the virus. COVID19-PEB could be developed as a rapid and accurate diagnostic tool for COVID-19.

An Antibody-Immobilized Silica Inverse Opal Nanostructure for Label-Free Optical Biosensors.

Three-dimensional SiO2-based inverse opal (SiO2-IO) nanostructures were prepared for use as biosensors. SiO2-IO was fabricated by vertical deposition and calcination processes. Antibodies were immobilized on the surface of SiO2-IO using 3-aminopropyl trimethoxysilane (APTMS), a succinimidyl-[(N-maleimidopropionamido)-tetraethyleneglycol] ester (NHS-PEG4-maleimide) cross-linker, and protein G. The highly accessible surface and porous structure of SiO2-IO were beneficial for capturing influenza viruses on the antibody-immobilized surfaces. Moreover, as the binding leads to the redshift of the reflectance peak, the influenza virus could be detected by simply monitoring the change in the reflectance spectrum without labeling. SiO2-IO showed high sensitivity in the range of 10³-105 plaque forming unit (PFU) and high specificity to the influenza A (H1N1) virus. Due to its structural and optical properties, SiO2-IO is a promising material for the detection of the influenza virus. Our study provides a generalized sensing platform for biohazards as various sensing strategies can be employed through the surface functionalization of three-dimensional nanostructures.

On-Site Detection of Aflatoxin B1 in Grains by a Palm-Sized Surface Plasmon Resonance Sensor.

Aflatoxins (AFs) are highly toxic compounds that can cause both acute and chronic toxicity in humans. Aflatoxin B1 (AFB1) is considered the most toxic of AFs. Therefore, the rapid and on-site detection of AFB1 is critical for food safety management. Here, we report the on-site detection of AFB1 in grains by a portable surface plasmon resonance (SPR) sensor. For the detection of AFB1, the surface of an SPR Au chip was sequentially modified by cysteine-protein G, AFB1 antibody, and bovine serum albumin (BSA). Then, the sample solution and AFB1-BSA conjugate were flowed onto the Au chip in serial order. In the absence of AFB1, the SPR response greatly increased due to the binding of AFB1-BSA on the Au chip. In the presence of AFB1, the SPR response showed little change because the small AFB1 molecule binds on the Au chip instead of the large AFB1-BSA molecule. By using this portable SPR-based competitive immunoassay, the sensor showed low limits of detection (2.51 ppb) and quantification (16.32 ppb). Furthermore, we successfully detected AFB1 in rice, peanut, and almond samples, which suggests that the proposed sensing method can potentially be applied to the on-site monitoring of mycotoxins in food.

Simple and rapid detection of bacteria using a nuclease-responsive DNA probe.

We demonstrate simple and rapid bacterial detection using a nuclease-responsive DNA probe. The probe consisting of a fluorescent dye and a quencher at the 5' and 3' termini, respectively, was designed to be cleaved by nucleases such as endonucleases, exonucleases, and DNases, which are released from bacteria using an optimized lysis buffer. The fluorescence signal of the cleaved DNA probe correlates with the number of Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus, and the detection limit was 103 CFU for E. coli and 104 CFU for S. aureus. Moreover, this method is specific for live bacteria and takes just one minute to get the signal including sample collection. These features make the present bacterial detection method a powerful on-site bacterial contamination assay which is simple, rapid, and quantitative.

A Multivalent Structure-Specific RNA Binder with Extremely Stable Target Binding but Reduced Interaction with Nonspecific RNAs.

By greatly enhancing binding affinities against target biomolecules, multivalent interactions provide an attractive strategy for biosensing. However, there is also a major concern for increased binding to nonspecific targets by multivalent binding. A range of charge-engineered probes of a structure-specific RNA binding protein PAZ as well as multivalent forms of these PAZ probes were constructed by using diverse multivalent avidin proteins (2-mer, 4-mer, and 24-mer). Increased valency vastly enhanced the binding stability of PAZ to structured target RNA. Surprisingly, nonspecific RNA binding of multivalent PAZ can be reduced even below that of the PAZ monomer by controlling negative charges on both PAZ and multivalent avidin scaffolds. The optimized 24-meric PAZ showed nearly irreversible binding to target RNA with negligible binding to nonspecific RNA, and this ultra-specific 24-meric PAZ probe allowed SERS detection of intact microRNAs at an attomolar level.

A novel and highly specific phage endolysin cell wall binding domain for detection of Bacillus cereus.

Rapid, specific and sensitive detection of pathogenic bacteria is crucial for public health and safety. Bacillus cereus is harmful as it causes foodborne illness and a number of systemic and local infections. We report a novel phage endolysin cell wall-binding domain (CBD) for B. cereus and the development of a highly specific and sensitive surface plasmon resonance (SPR)-based B. cereus detection method using the CBD. The newly discovered CBD from endolysin of PBC1, a B. cereus-specific bacteriophage, provides high specificity and binding capacity to B. cereus. By using the CBD-modified SPR chips, B. cereus can be detected at the range of 10(5)-10(8) CFU/ml. More importantly, the detection limit can be improved to 10(2) CFU/ml by using a subtractive inhibition assay based on the pre-incubation of B. cereus and CBDs, removal of CBD-bound B. cereus, and SPR detection of the unbound CBDs. The present study suggests that the small and genetically engineered CBDs can be promising biological probes for B. cereus. We anticipate that the CBD-based SPR-sensing methods will be useful for the sensitive, selective, and rapid detection of B. cereus.

Composition-selective fabrication of ordered intermetallic Au-Cu nanowires and their application to nano-size electrochemical glucose detection.

Bimetallic nanostructures can provide distinct and improved physicochemical properties by the coupling effect of the two metal components, making them promising materials for a variety of applications. Herein, we report composition-selective fabrication of ordered intermetallic Au-Cu nanowires (NWs) by two-step chemical vapor transport method and their application to nano-electrocatalytic glucose detection. Ordered intermetallic Au3Cu and AuCu3 NWs are topotaxially fabricated by supplying Cu-containing chemicals to pre-synthesized single-crystalline Au NW arrays. The composition of fabricated Au-Cu NWs can be selected by changing the concentration of Cu-containing species. Interestingly, Au3Cu NW electrodes show unique electrocatalytic activity for glucose oxidation, allowing us to detect glucose without interference from ascorbic acid. Such interference-free detection of glucose is attributed to the synergistic effect, induced by incorporation of Cu in Au. We anticipate that Au3Cu NWs could show possibility as efficient nano-size electrochemical glucose sensors and the present fabrication method can be employed to fabricate valuable ordered intermetallic nanostructures.

Ultra-specific zeptomole microRNA detection by plasmonic nanowire interstice sensor with Bi-temperature hybridization.

MicroRNAs (miRNAs) are emerging new biomarkers for many human diseases. To fully employ miRNAs as biomarkers for clinical diagnosis, it is most desirable to accurately determine the expression patterns of miRNAs. The optimum miRNA profiling method would feature 1) highest sensitivity with a wide dynamic range for accurate expression patterns, 2) supreme specificity to discriminate single nucleotide polymorphisms (SNPs), and 3) simple sensing processes to minimize measurement variation. Here, an ultra-specific detection method of miRNAs with zeptomole sensitivity is reported by applying bi-temperature hybridizations on single-crystalline plasmonic nanowire interstice (PNI) sensors. This method shows near-perfect accuracy of SNPs and a very low detection limit of 100 am (50 zeptomole) without any amplification or labeling steps. Furthermore, multiplex sensing capability and wide dynamic ranges (100 am-100 pm) of this method allows reliable observation of the expression patterns of miRNAs extracted from human tissues. The PNI sensor offers combination of ultra-specificity and zeptomole sensitivity while requiring two steps of hybridization between short oligonucleotides, which could present the best set of features for optimum miRNA sensing method.

Electrotriggered, spatioselective, quantitative gene delivery into a single cell nucleus by Au nanowire nanoinjector.

Delivery of bioactive materials into a cell is highly important in the study of cell biology and medical treatments. Ideal nanoinjectors should be able to deliver biomaterials with high spatial resolution while causing minimum cell damage. We developed a Au nanowire (NW) nanoinjector that has the thinnest diameter (100­150 nm) among the DNA delivering devices as well as optimum mechanical properties, minimizing cell damage. Well-defined (111) single-crystalline Au surface and high electric conductivity of a Au NW nanoinjector allow precisely timed and efficient electrochemical release of DNA molecules attached on a Au NW surface. Both linear DNA and plasmid DNA were delivered separately and showed successful expression. The Au NW nanoinjector would find important biomedical applications in the fields such as gene therapy, DNA vaccination, targeted drug delivery, and probe/control of cell signaling events.

PoreGlow: A split green fluorescent protein-based system for rapid detection of Listeria monocytogenes.

Listeria monocytogenes, a severe foodborne pathogen causing severe diseases underscores the necessity for the development of a detection system with high specificity, sensitivity and utility. Herein, the PoreGlow system, based on split green fluorescent protein (GFP), was developed and assessed for the fast and accurate detection of L. monocytogenes. Split GFP-encapsulated liposomes were optimized for targeted analysis. The system utilizes listeriolysin O (LLO), a toxin produced by L. monocytogenes that enlarges the pores split GFP-encapsulated liposomes, to detect L. monocytogenes by measuring the fluorescent signal generated when the encapsulated GFP is released and reacted with the externally added fragment of the split GFP. The system exhibited a limit of detection of 0.17 µg/ml for LLO toxin and 10 CFU/mL for L. monocytogenes with high sensitivity and specificity and no cross-reactivity with other bacteria. The PoreGlow system is practical, rapid, and does not require sample pre-treatment, making it a promising tool for the early detection of L. monocytogenes in food products, which is crucial for preventing outbreaks and protecting public health.

A CRISPR/Cas12 trans-cleavage reporter enabling label-free colorimetric detection of SARS-CoV-2 and its variants.

We present a label-free colorimetric CRISPR/Cas-based method enabling affordable molecular diagnostics for SARS-CoV-2. This technique utilizes 3,3'-diethylthiadicarbocyanine iodide (DISC2(5)) which exhibits a distinct color transition from purple to blue when it forms dimers by inserting into the duplex of the thymidine adenine (TA) repeat sequence. Loop-mediated isothermal amplification (LAMP) or recombinase polymerase amplification (RPA) was used to amplify target samples, which were subsequently subjected to the CRISPR/Cas12a system. The target amplicons would activate Cas12a to degrade nearby TA repeat sequences, preserving DISC2(5) in its free form to display purple as opposed to blue in the absence of the target. Based on this design approach, SARS-CoV-2 RNA was colorimetrically detected very sensitively down to 2 copies/µL, and delta and omicron variants of SARS-CoV-2 were also successfully identified. The practical diagnostic utility of this method was further validated by reliably identifying 179 clinical samples including 20 variant samples with 100% clinical sensitivity and specificity. This technique has the potential to become a promising CRISPR-based colorimetric platform for molecular diagnostics of a wide range of target pathogens.

CRISPR/Cas12a antifouling nanocomposite electrochemical biosensors enable amplification-free detection of Monkeypox virus in complex biological fluids.

The escalating global threat of infectious diseases, including monkeypox virus (MPXV), necessitates advancements in point-of-care diagnostics, moving beyond the constraints of conventional methods tethered to centralized laboratories. Here, we introduce multiple CRISPR RNA (crRNA)-based biosensors that can directly detect MPXV within 35 minutes without pre-amplification, leveraging the enhanced sensitivity and antifouling attributes of the BSA-based nanocomposite. Multiple crRNAs, strategically targeting diverse regions of the F3L gene of MPXV, are designed and combined to amplify Cas12a activation and its collateral cleavage of reporter probes. Notably, our electrochemical sensors exhibit the detection limit of 669 fM F3L gene without amplification, which is approximately a 15-fold improvement compared to fluorescence detection. This sensor also shows negligible changes in peak current after exposure to complex biological fluids, such as whole blood and serum, maintaining its sensitivity at 682 fM. This sensitivity is nearly identical to the conditions when only the F3L gene was present in PBS. In summary, our CRISPR-based electrochemical biosensors can be utilized as a high-performance diagnostic tool in resource-limited settings, representing a transformative leap forward in point-of-care testing. Beyond infectious diseases, the implications of this technology extend to various molecular diagnostics, establishing itself as a rapid, accurate, and versatile platform for detection of target analytes.

Multifunctional self-priming hairpin probe-based isothermal nucleic acid amplification and its applications for COVID-19 diagnosis.

We herein present a multifunctional self-priming hairpin probe-based isothermal amplification, termed MSH, enabling one-pot detection of target nucleic acids. The sophisticatedly designed multifunctional self-priming hairpin (MSH) probe recognizes the target and rearranges to prime itself, triggering the amplification reaction powered by the continuously repeated extension, nicking, and target recycling. As a consequence, a large number of double-stranded DNA (dsDNA) amplicons are produced that could be monitored in real-time using a dsDNA-intercalating dye. Based on this unique design approach, the nucleocapsid (N) and the open reading frame 1 ab (ORF1ab) genes of SARS-CoV-2 were successfully detected down to 1.664 fM and 0.770 fM, respectively. The practical applicability of our method was validated by accurately diagnosing 60 clinical samples with 93.33% sensitivity and 96.67% specificity. This isothermal one-pot MSH technique holds great promise as a point-of-care testing protocol for the reliable detection of a wide spectrum of pathogens, particularly in resource-limited settings.