Lateral Flow Test Strip Research Articles

Rapid Detection of Alpha-Fetoprotein (AFP) with Lateral Flow Aptasensor

We present a lateral flow aptasensor for the visual detection of alpha-fetoprotein (AFP) in human serum. Leveraging the precise molecular recognition capabilities of aptamers and the distinct optical features of gold nanoparticles, a model system utilizing AFP as the target analyte, along with a pair of aptamer probes, is implemented to establish proof-of-concept on standard lateral flow test strips. It is the first report of an antibody-free lateral flow assay using aptamers as recognition probes for the detection of AFP. The analysis circumvents the numerous incubation and washing steps that are typically involved in most current aptamer-based protein assays. Qualitative analysis involves observing color changes in the test area, while quantitative data are obtained by measuring the optical response in the test zone using a portable strip reader. The biosensor exhibits a linear detection range for AFP concentrations between 10 and 100 ng/mL, with a minimum detection limit of 10 ng/mL. Additionally, it has been successfully applied to detect AFP in human serum samples. The use of aptamer-functionalized gold nanoparticle probes in a lateral flow assay offers great promise for point-of-care applications and fast, on-site detection.

Paper-Based Aptasensor Assay for Detection of Food Adulterant Sildenafil.

Sildenafil is used to treat erectile dysfunction and pulmonary arterial hypertension but is often illicitly added to energy drinks and chocolates. This study introduces a lateral flow strip test using aptamers specific to sildenafil for detecting its illegal presence in food. The process involved using graphene oxide SELEX to identify high-affinity aptamers, which were then converted into molecular gate structures on mesoporous silica nanoparticles, creating a unique signaling system. This system was integrated into lateral flow chromatography strips and tested on buffers and chocolate samples containing sildenafil. The method simplifies the lateral flow assay (LFA) for small molecules and provides a tool for signal amplification. The detection limit for these strips was found to be 68.2 nM (31.8 µg/kg) in spiked food samples.

A smartphone-integrated rapid and sensitive lateral flow test strip for quantitative detection of Fusarium spp. in maize samples

A smartphone-integrated rapid and sensitive lateral flow test strip for quantitative detection of Fusarium spp. in maize samples

G-quadruplex fluorescent and lateral flow colorimetric aptasensor for the detection of capsaicin in illicit cooking oil

The safety of cooking oil is a major global concern and capsaicin (CAP) serves as a crucial marker for assessing its quality. To address this issue, we have devised an innovative biosensor using a truncated aptamer, Cap-1–2, as the foundation for a dual-mode aptasensor. The aptasensor employs a combination of Mg2+-dependent DNAzyme (MNAzyme) cleavage and hybridization chain reaction (HCR) to achieve highly sensitive CAP detection. Notably, the fluorescence signal originates from the inherent fluorescence of thioflavin T (ThT) embedded in a G-quadruplex (G4) structure, enabling the detection of CAP in high-throughput oil samples. Additionally, the biosensor employed the MNAzyme to cleave biotinylated DNA (S1-Biotin), which is subsequently ligated to functionalized gold nanoparticles (AuNPs-Poly A-cDNA), generating outstanding colorimetric signals on lateral flow test strips for immediate point-of-care testing (POCT). The linear detection ranges of the established fluorescence and colorimetric aptasensor were 0.15–80 ng mL−1 and 0.2–100 ng mL−1, respectively, and the limits of detection (LOD) were 0.054 ng mL−1 and 0.137 ng mL−1, respectively. These results demonstrate the practicality of the aptasensor for detecting CAP in real-world samples. Moreover, the aptasensor surpasses traditional biosensors in terms of high-throughput, portability, and sensitivity, making it highly promising for rapid detection in the food industry.

Preliminary Development of DNA Aptamer Quantum Dot-Based Competitive Lateral Flow Assays for Saxitoxin and Tetrodotoxin.

Initial proof-of-concept development of competitive lateral flow (LF) test strips involving red quantum dots (Qdots) is demonstrated with visible confirmation of the competitive displacement of Qdot-protein-saxitoxin (Stx) and -tetrodotoxin (Ttx) conjugates over 15min. The measured limits of detection (LODs) for the best versions of the assays were in the 1-2µg range when a bovine serum albumin (BSA)-Stx or -Ttx conjugate was used as the analyte for safety reasons and NIH ImageJ image analysis was applied. However, when one assumes that only one of the primary amines in BSA labeled with Stx or Ttx is needed as the "epitope" for capture and calculates the weight of the toxin adducts on an equal mole basis to that of 1µg of toxin-BSA conjugate, the LODs are as low as 4.5ng of toxin. No cross-reactivity was seen for these prototype test strips when BSA-Stx and BSA-Ttx were switched as analytes at or near their respective LODs which is in agreement with published specificity and affinity studies for these aptamers in the literature.

CRISPR Diagnostics for Quantification and Rapid Diagnosis of Myotonic Dystrophy Type 1 Repeat Expansion Disorders.

Repeat expansion disorders, exemplified by myotonic dystrophy type 1 (DM1), present challenges in diagnostic quantification because of the variability and complexity of repeat lengths. Traditional diagnostic methods, including PCR and Southern blotting, exhibit limitations in sensitivity and specificity, necessitating the development of innovative approaches for precise and rapid diagnosis. Here, we introduce a CRISPR-based diagnostic method, REPLICA (repeat-primed locating of inherited disease by Cas3), for the quantification and rapid diagnosis of DM1. This method, using in vitro-assembled CRISPR-Cas3, demonstrates superior sensitivity and specificity in quantifying CTG repeat expansion lengths, correlated with disease severity. We also validate the robustness and accuracy of CRISPR diagnostics in quantitatively diagnosing DM1 using patient genomes. Furthermore, we optimize a REPLICA-based assay for point-of-care-testing using lateral flow test strips, facilitating rapid screening and detection. In summary, REPLICA-based CRISPR diagnostics offer precise and rapid detection of repeat expansion disorders, promising personalized treatment strategies.

A novel lateral flow assay for lead ion detection based on G-quadruplex

Lead ion residues pose potential health risks to humans. To develop a cost-effective and user-friendly portable lead ion detection method, a novel lateral flow test strip was designed and fabricated based on the G-quadruplex structure. Initially, G4 and its complementary strand antiG4 are in a double-stranded form in the assay solution. After adding lead ions, these ions compete with antiG4 to bind to G4 and form G-quadruplex structures, thus releasing a large number of antiG4 single strands. Through base pairing, one end of the antiG4 was linked to a recognition element containing gold nanoparticles, while the other end was captured by a test line probe, resulting in a red band on the test line. The color change of the test line was positively correlated with the concentration of lead ions. Results showed that by observing the color change of the test line under optimized experimental conditions, lead ion concentration could be detected with a visual detection limit of 20 nM. Quantitative analysis using ImageJ software indicated that the test strip had a linear detection range of 10–2000 nM, with a detection limit of 7.32 nM and significant specificity. The recovery rate in bottled drinking water ranged from 91.19 % to 126.04 %, providing a portable and simple new method for on-site detection of residual lead ions in water environments.

M13mp18 nanoscaffold-based strip sensor for detecting influenza A virus (H1N1)

We report the development of an amplification-free nucleic acid assay using lateral flow test strips. M13mp18 nanoscaffold signal amplification permitted the visual detection of H1N1 RNA in 20 min. M13mp18 nanoscaffold supported a set of backbone, variable, detection, and modified oligonucleotides. A bridging-type FAM probe/H1N1 RNA/M13mp18 was used in the lateral flow test strip format. The bridging-type complexes were captured on the test zone. The lower limit of detection of specific H1N1 RNA was 1 pM. The method directly detected H1N1 RNA without amplification, eliminating the risk of amplicon contamination and lessening the chance of false positives. This biosensor has potential value for field testing in resource-limited settings and monitoring viral loads in recovering patients. The amplification-free lateral flow detection of RNA provides an alternative between amplification-based RNA assays and protein antigen assays for facilitating the rapid identification of H1N1 virus during point-of-care testing.

Development of a Recombinase Polymerase Amplification-Coupled CRISPR/Cas12a Platform for Rapid Detection of Antimicrobial-Resistant Genes in Carbapenem-Resistant Enterobacterales.

The worldwide spread of carbapenemase-producing Enterobacterales (CPE) represents a significant threat owing to the high mortality and morbidity rates. Traditional diagnostic methods are often too slow and complex for rapid point-of-care testing. Therefore, we developed a recombinase polymerase amplification (RPA)-coupled CRISPR/Cas12a system (RCCS), a rapid, accurate, and simple diagnostic platform for detecting antimicrobial-resistant genes. The RCCS detected carbapenemase genes (blaKPC and blaNDM) within 50 min, including 10 min for DNA extraction and 30-40 min for RCCS reaction (a 20 min RPA reaction with a 10-20-min CRISPR/Cas12a assay). Fluorescence signals obtained from the RCCS platform were visualized using lateral-flow test strips (LFSs) and real-time and endpoint fluorescence. The LFS clearly displayed test lines while detecting carbapenemase genes. Furthermore, the RCCS platform demonstrated high sensitivity by successfully detecting blaKPC and blaNDM at the attomolar and picomolar levels, respectively. The accuracy of the RCCS platform was validated with clinical isolates of Klebsiella pneumoniae and Escherichia coli; a 100% detection accuracy was achieved, which has not been reported when using conventional PCR. Overall, these findings indicate that the RCCS platform is a powerful tool for rapid and reliable detection of carbapenemase-encoding genes, with significant potential for implementation in point-of-care settings and resource-limited environments.

Strand displacement amplification combining photothermal-colorimetric dual mode test strip for ultra-sensitive detection of patulin

Patulin (PAT) is a fungal toxin often found in fruit and juice products. The sensitive and fast detection of PAT can help the control of PAT-induced health risk. However, the lack of efficient and stable antibody for PAT hindered the advancement of its test strips. Here, we constructed a nucleic acid-based lateral flow test strip (NALFTS) and used photothermal-colorimetric dual mode for the detection of PAT. The binding of PAT in samples to the aptamer on magnetic separation probe MNPs-Apt@cDNA induced the release of cDNA competitively. Subsequently, cDNA initiates strand displacement amplification (SDA), generating numerous single-strand DNA (ssDNA). These ssDNA molecules were analyzed by a test strip with photothermal-colorimetric dual signal. The developed method demonstrated a limit of detection (LOD) of 5 pg mL−1 and a quantitative range of 0.01 ∼ 320 ng mL−1 in photothermal mode, and a LOD of 0.1 ng mL−1 and a quantitative range of 0.1 ∼ 40 ng mL−1 in colorimetric mode. This SDA-NALFTS offered dual signals corroborating each other to provide more reliable results for rapid screening or accurate monitoring of PAT in juice.

Establishment of a platform based on dual RPA combined with CRISPR/Cas12a for the detection of Klebsiella pneumoniae and its KPC resistance gene.

Carbapenem resistant Klebsiella pneumoniae (CRKP) can cause serious hospital- and community-acquired infections. Treatment for CRKP infection is limited, resulting in prolonged hospitalization and high consultation costs. The KPC genotype has the highest detection rate of CRKP, and its mortality rate is higher than the overall mortality rate of CRKP. However, traditional testing methods have disadvantages such as long time and reliance on complex and sophisticated instruments, which are not conducive to rapid screening for CRKP. Therefore, this study aimed to establish a detection platform for early screening of CRKP so that effective antimicrobial therapy could be administered promptly to prevent the widespread spread of CRKP. We integrated dual RPA with CRISPR/Cas12a to establish a dual platform for the detection of K. pneumoniae (Kp) rcsA-specific gene and KPC resistance gene. Four result reading methods were established, including fluorescence detection (FD), blue light irradiation detection (BLID), ultraviolet irradiation detection (UID), and lateral flow test strips (LFTS). For the rcsA gene, the LOD of FD was 1 × 10pg/μL, and the other three methods could detect 1 × 101pg/μL of bacterial DNA. As for the KPC gene, four resultant readout methods were able to detect 1 × 102pg/μL of bacterial DNA. In 59 clinical strains tested, the dual RPA-CRISPR/Cas12a detection of the rcsA had 100% sensitivity, specificity, and accuracy compared to the culture method. Compared with the drug sensitivity test, the sensitivity of dual RPA-CRISPR/Cas12a detection for the KPC was 85.71%, the specificity was 100%, and the accuracy was 94.92%. In summary, our dual RPA-CRISPR/Cas12a platform proved to be rapid, precise, and convenient for the efficient detection of Kp with KPC in the laboratory or at the point of care.

Freeze-Driven Adsorption of Oligonucleotides with polyA-Anchors on Au@Pt Nanozyme.

A promising and sought-after class of nanozymes for various applications is Pt-containing nanozymes, primarily Au@Pt, due to their easy preparation and remarkable catalytic properties. This study aimed to explore the freeze-thaw method for functionalizing Pt-containing nanozymes with oligonucleotides featuring a polyadenine anchor. Spherical gold nanoparticles ([Au]NPs) were synthesized and subsequently used as seeds to produce urchin-like Au@Pt nanoparticles ([Au@Pt]NPs) with an average diameter of 29.8 nm. The nanoparticles were conjugated with a series of non-thiolated DNA oligonucleotides, each consisting of three parts: a 5'-polyadenine anchor (An, with n = 3, 5, 7, 10; triple-branched A3, or triple-branched A5), a random sequence of 23 nucleotides, and a linear polyT block consisting of seven deoxythymine residues. The resulting conjugates were characterized using transmission electron microscopy, spectroscopy, dynamic light scattering, and emission detection of the fluorescent label at the 3'-end of each oligonucleotide. The stability of the conjugates was found to depend on the type of oligonucleotide, with decreased stability in the row of [Au@Pt]NP conjugates with A7 > A5 > 3A3 > 3A5 > A10 > A3 anchors. These [Au@Pt]NP-oligonucleotide conjugates were further evaluated using lateral flow test strips to assess fluorescein-specific binding and peroxidase-like catalytic activity. Conjugates with A3, A5, A7, and 3A3 anchors showed the highest levels of signals of bound labels on test strips, exceeding conjugates in sensitivity by up to nine times. These findings hold significant potential for broad application in bioanalytical systems.

Trimodal Multiplexed Lateral Flow Test Strips Assisted with a Portable Microfluidic Centrifugation Device.

During the COVID-19 pandemic, the use of lateral flow assays (LFAs) expanded significantly, offering testing beyond traditional health care. Their appeal lies in the ease of use, affordability, and quick results. However, LFAs often have lower sensitivity and specificity compared with ELISA and PCR tests. Efforts to improve LFAs have increased detection times and complexity, limiting their use in large-scale point-of-care settings. To address this, we propose a novel approach using probes that generate multiple signals to enhance the sensitivity and selectivity. This concept also allows multiplexed LFAs to detect multiple analytes concurrently. We developed a trimodal probe that integrates fluorescence, color, and magnetism into a single nanohybrid. The strong plasmonic absorption and high fluorescence of Au nanoparticles and polymer dots enable qualitative and semiquantitative diagnosis, while the magnetic signal facilitates accurate quantitative measurements. As proof-of-concept targets, we selected CYFRA 21-1 and CA15-3, biomarkers for lung and breast cancer, respectively. This trimodal LFA demonstrated a remarkable detection limit of 0.26 ng/mL for CYFRA 21-1 and 2.8 U/mL for CA15-3. To the best of our knowledge, this is the first platform of a trimodal LFA with multiplexing ability. The platform's accuracy and reliability were validated using clinical serum samples, showing excellent consistency with electrochemiluminescence immunoassay results. This universal concept can be applied to other targets, paving the way for the next-generation LFAs.

Microstereolithography-fabricated microneedles for fluid sampling of histamine-contaminated tuna

A custom-designed microneedle sampling system was prepared using dynamic mask microstereolithography; this sampling system was used for determination of histamine content in fresh, histamine-spiked, and spoiled tuna flesh. Lateral flow (test strip) assays were successfully utilized in the microneedle sampling system to assess histamine content. Good agreement was noted between data obtained from the microneedle sampling system and a commercially available histamine detection kit. A discrepancy was noted in the results from the microneedle sampling system and the commercially available histamine detection kit at low (negative) levels of histamine. There was an improvement in the agreement between the microneedle sampling system and the commercially available histamine detection kit at higher histamine levels. The results, which showed an improvement in the test duration and the amount of reagent needed for histamine detection, indicate the promise of printed microneedle sampling systems for histamine detection in seafood samples and other types of food testing.

A Glycoprotein-Based Surface-Enhanced Raman Spectroscopy-Lateral Flow Assay Method for Abrin and Ricin Detection.

Abrin and ricin, both type II ribosome-inactivating proteins, are toxins of significant concern and are under international restriction by the Chemical Weapons Convention and the Biological and Toxin Weapons Convention. The development of a rapid and sensitive detection method for these toxins is of the utmost importance for the first emergency response. Emerging rapid detection techniques, such as surface-enhanced Raman spectroscopy (SERS) and lateral flow assay (LFA), have garnered attention due to their high sensitivity, good selectivity, ease of operation, low cost, and disposability. In this work, we generated stable and high-affinity nanotags, via an efficient freezing method, to serve as the capture module for SERS-LFA. We then constructed a sandwich-style lateral flow test strip using a pair of glycoproteins, asialofetuin and concanavalin A, as the core affinity recognition molecules, capable of trace measurement for both abrin and ricin. The limit of detection for abrin and ricin was 0.1 and 0.3 ng/mL, respectively. This method was applied to analyze eight spiked white powder samples, one juice sample, and three actual botanic samples, aligning well with cytotoxicity assay outcomes. It demonstrated good inter-batch and intra-batch reproducibility among the test strips, and the detection could be completed within 15 min, indicating the suitability of this SERS-LFA method for the on-site rapid detection of abrin and ricin toxins.

CoHIT: a one-pot ultrasensitive ERA-CRISPR system for detecting multiple same-site indels

Genetic testing is crucial for precision cancer medicine. However, detecting multiple same-site insertions or deletions (indels) is challenging. Here, we introduce CoHIT (Cas12a-based One-for-all High-speed Isothermal Test), a one-pot CRISPR-based assay for indel detection. Leveraging an engineered AsCas12a protein variant with high mismatch tolerance and broad PAM scope, CoHIT can use a single crRNA to detect multiple NPM1 gene c.863_864 4-bp insertions in acute myeloid leukemia (AML). After optimizing multiple parameters, CoHIT achieves a detection limit of 0.01% and rapid results within 30 minutes, without wild-type cross-reactivity. It successfully identifies NPM1 mutations in 30 out of 108 AML patients and demonstrates potential in monitoring minimal residual disease (MRD) through continuous sample analysis from three patients. The CoHIT method is also competent for detecting indels of KIT, BRAF, and EGFR genes. Integration with lateral flow test strips and microfluidic chips highlights CoHIT’s adaptability and multiplexing capability, promising significant advancements in clinical cancer diagnostics.

Introducing Triplex Forming Oligonucleotide into Loop-Mediated Isothermal Amplification for Developing a Lateral Flow Biosensor for Streptococci Detection.

Loop-mediated isothermal amplification (LAMP) technology is extensively utilized for the detection of infectious diseases owing to its rapid processing and high sensitivity. Nevertheless, conventional LAMP signaling methods frequently suffer from a lack of sequence specificity. This study integrates a triplex-forming oligonucleotide (TFO) probe into the LAMP process to enhance sequence specificity. This TFO-LAMP technique was applied for the detection of Group B Streptococcus (GBS). The TFO probe is designed to recognize a specific DNA sequence, termed the TFO targeting sequence (TTS), within the amplified product, facilitating detection via fluorescent instrumentation or lateral flow biosensors. A screening method was developed to identify TFO sequences with high affinity to integrate TFO into LAMP, subsequently incorporating a selected TTS into an LAMP primer. In the TFO-LAMP assay, a FAM-labeled TFO is added to target the TTS. This TFO can be captured by an anti-FAM antibody on lateral flow test strips, thus creating a nucleic acid testing biosensor. The efficacy of the TFO-LAMP assay was confirmed through experiments with specimens spiked with varying concentrations of GBS, demonstrating 85% sensitivity at 300 copies and 100% sensitivity at 30,000 copies. In conclusion, this study has successfully developed a TFO-LAMP technology that offers applicability in lateral flow biosensors and potentially other biosensor platforms.

Proteolytic reaction-based versatile electrochemical biosensor chip for simple and quantitative point-of-care testing

Electrochemical protease biosensors are well-suited for simple and quantitative point-of-care testing. However, there has been limited development of versatile chip formats similar to lateral-flow and glucose test strips for electrochemical proteases biosensors. This paper presents a versatile electrochemical biosensor chip for the wash-free and sensitive detection of proteases. The chip comprises (i) a reaction well, (ii) micropatterned electrodes on a glass substrate, and (iii) target protease-dependent filter paper. All (bio)chemicals required for the proteolytic reaction and signal amplification were predried on filter paper, which was then placed inside the reaction well. Upon injecting the sample solution into the reaction well, most of the dried (bio)chemicals dissolved rapidly. Wash-free and sensitive protease detection was achieved using electrochemical-enzymatic (EN) redox cycling of a redox-active species (4-amino-1-naphthol, AN) liberated from a peptide substrate through a rapid proteolytic reaction. The EN redox cycling involves a working electrode, AN, glucose dehydrogenase, and glucose. To enhance dissolvability and stability, (bio)chemicals were dried using a surfactant and stabilizer and stored at 4 °C with vacuum sealing. To minimize the effect of background variation among the different samples, a high concentration of glucose (200 mM) was used, and the difference between the two charge values obtained before and after the incubation period was measured. The biosensor chips successfully detected three proteases: thrombin, leucine aminopeptidase (LAP), and 3CL protease from coronavirus SARS-CoV-2 (Mpro). The detection limits of thrombin in artificial serum, Mpro in artificial saliva, and LAP in human serum were approximately 100, 100 pg/mL, and 8 U/L, respectively.

A lateral flow assay for miRNA-21 based on CRISPR/Cas13a and MnO2 nanosheets-mediated recognition and signal amplification.

The point-of-care testing (POCT) of miRNA has significant application in medical diagnosis, yet presents challenges due to their characteristics of high homology, low abundance, and short length, which hinders the achievement of quick detection with high specificity and sensitivity. In this study, a lateral flow assay based on the CRISPR/Cas13a system and MnO2 nanozyme was developed for highlysensitive detection of microRNA-21 (miR-21). The CRISPR/Cas13a cleavage system exhibits the ability to recognize the specific oligonucleotide sequence, where two-base mismatches significantly impact the cleavage activity of the Cas13a. Upon binding of the target to crRNA, the cleavage activity of Cas13a is activated, resulting in the unlocking of the sequence and initiating strand displacement, thereby enabling signal amplification to produce a new sequence P1. When applying the reaction solution to the lateral flow test strip, P1 mediates the capture of MnO2 nanosheets (MnO2 NSs) on the T zone, which catalyzes the oxidation of the pre-immobilized colorless substrate 3,3',5,5'-tetramethylbenzidine (TMB) on the T zone and generates the blue-green product (ox-TMB). The change in gray value is directly proportional to the concentration of miR-21, allowing for qualitative detection through visual inspection and quantitative measurement using ImageJ software. This method achieves the detection of miR-21 within a rapid 10-min timeframe, and the limit of detection (LOD) is 0.33pM. With the advantages of high specificity, simplicity, and sensitivity, the lateral flow test strip and the design strategy hold great potential for the early diagnosis of related diseases.

The Development of Aptamer-Based Gold Nanoparticle Lateral Flow Test Strips for the Detection of SARS-CoV-2 S Proteins on the Surface of Cold-Chain Food Packaging.

The COVID-19 pandemic over recent years has shown a great need for the rapid, low-cost, and on-site detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this study, an aptamer-based colloidal gold nanoparticle lateral flow test strip was well developed to realize the visual detection of wild-type SARS-CoV-2 spike proteins (SPs) and multiple variants. Under the optimal reaction conditions, a low detection limit of SARS-CoV-2 S proteins of 0.68 nM was acquired, and the actual detection recovery was 83.3% to 108.8% for real-world samples. This suggests a potential tool for the prompt detection of SARS-CoV-2 with good sensitivity and accuracy, and a new method for the development of alternative antibody test strips for the detection of other viral targets.