A Highly Sensitive XNA-Based RT-qPCR Assay for the Identification of ALK, RET, and ROS1 Fusions in Lung Cancer.

Lung cancer is often triggered by genetic alterations that result in the expression of oncogenic tyrosine kinases. Specifically, ALK, RET, and ROS1 chimeric receptor tyrosine kinases are observed in approximately 5-7%, 1-2%, and 1-2% of NSCLC patients, respectively. The presence of these fusion genes determines the response to tyrosine kinase inhibitors. Thus, accurate detection of these gene fusions is essential in cancer research and precision oncology. To address this need, we have developed a multiplexed RT-qPCR assay using xeno nucleic acid (XNA) molecular clamping technology to detect lung cancer fusions. This assay can quantitatively detect thirteen ALK, seven ROS1, and seven RET gene fusions in FFPE samples. The sensitivity of the assay was established at a limit of detection of 50 copies of the synthetic template. Our assay has successfully identified all fusion transcripts using 50 ng of RNA from both reference FFPE samples and cell lines. After validation, a total of 77 lung cancer patient FFPE samples were tested, demonstrating the effectiveness of the XNA-based fusion gene assay with clinical samples. Importantly, this assay is adaptable to highly degraded RNA samples with low input amounts. Future steps involve expanding the testing to include a broader range of clinical samples as well as cell-free RNAs to further validate its applicability and reliability.

A new testing platform using fingerstick blood for quantitative antibody response evaluation after SARS-CoV-2 vaccination.

Testing and vaccination have been major components of the strategy for combating the ongoing COVID-19 pandemic. In this study, we have developed a quantitative anti-SARS-CoV-2 spike (S1) IgG antibody assay using a fingerstick dried blood sample. We evaluated the feasibility of using this high-throughput and quantitative anti-SARS-CoV-2 spike (S1) IgG antibody testing assay in vaccinated individuals. Fingerstick blood samples were collected and analyzed from 137 volunteers before and after receiving the Moderna or Pfizer mRNA vaccine. Anti-SARS-CoV-2 S1 IgG antibody could not be detected within the first 7 days after receiving the first vaccine dose, however, the assay reliably detected antibodies from day 14 onwards. In addition, no anti-SARS-CoV-2 nucleocapsid (N) protein IgG antibody was detected in any of the vaccinated or healthy participants, indicating that the anti-SARS-CoV-2 S1 IgG assay is specific for the mRNA vaccine-induced antibodies. The S1 IgG levels detected in fingerstick samples correlated with the levels found in venous blood plasma samples and with the efficacy of venous blood plasma samples in the plaque reduction neutralization test (PRNT). The assay displayed a limit of quantification (LOQ) of 0.59 µg/mL and was found to be linear in the range of 0.51-1000 µg/mL. Finally, its clinical performance displayed a Positive Percent Agreement (PPA) of 100% (95% CI: 0.89-1.00) and a Negative Percent Agreement (NPA) of 100% (95% CI: 0.93-1.00). In summary, the assay described here represents a sensitive, precise, accurate, and simple method for the quantitative detection and monitoring of post-vaccination anti-SARS-CoV-2 spike IgG responses.

A novel xenonucleic acid-mediated molecular clamping technology for early colorectal cancer screening.

BACKGROUND: Colorectal cancer (CRC) is one of the leading causes of cancer-related death. Early detection is critical to reduce CRC morbidity and mortality. In order to meet this need, we developed a molecular clamping assay called the ColoScape TM assay for early colorectal cancer diagnostics. METHODS: Nineteen mutations in four genes (APC, KRAS, BRAF and CTNNB1) associated with early events in CRC pathogenesis are targeted in the ColoScapeTM assay. Xenonucleic Acid (XNA)-mediated qPCR clamping technology was applied to minimize the wild-type background amplification in order to improve assay sensitivity of CRC mutation detection. The assay analytical performance was verified and validated, cfDNA and FFPE CRC patient samples were evaluated, and an ROC curve was applied to evaluate its performance. RESULTS: The data showed that the assay analytical sensitivity was 0.5% Variant Allele Frequency, corresponding to ~7-8 copies of mutant DNA with 5 ng total DNA input per test. This assay is highly reproducible with intra-assay CV of <3% and inter-assay CV of <5%. We have investigated 380 clinical samples including plasma cfDNA and FFPE samples from patients with precancerous and different stages of CRC. The preliminary assay clinical specificity and sensitivity for CRC cfDNA were: 100% (95% CI, 80.3-97.5%) and 92.2% (95% CI, 94.7-100%), respectively, with AUC of 0.96; 96% specificity (95% CI, 77.6-99.7%) and 92% sensitivity (95% CI, 86.1-95.6%) with AUC of 0.94 for CRC FFPE; 95% specificity (95% CI, 82.5%-99.1%) and 62.5% sensitivity (95% CI, 35.8%-83.7%) with AUC of 0.79 for precancerous lesions cfDNA. CONCLUSIONS: The XNA-mediated molecular clamping assay is a rapid, precise, and sensitive assay for the detection of precancerous lesions cfDNA and CRC cfDNA or FFPE samples.

A high-throughput microsphere-based immunoassay of anti-SARS-CoV-2 IgM testing for COVID-19 diagnostics.

The pandemic of novel coronavirus disease COVID-19 is rapidly expanding across the world. A positive result of antibody tests suggests that the individual has potentially been exposed to SARS-CoV-2, thus allowing to identify asymptomatic infections and determine the seroprevalence in a given population. The aim of this study was to evaluate the performances of a newly developed high throughput immunoassay for anti-SARS-CoV-2 IgM antibody detection on the Luminex MAGPIX platform. Clinical agreement studies were performed in 42 COVID-19 patient serum samples and 162 negative donor serum/plasma samples. Positive percent agreement (PPA) was 42.86% (95% CI: 9.90% to 81.59%), 71.43% (95% CI: 29.04% to 96.33%), and 28.57% (95% CI: 13.22% to 48.67%) for samples collected on 0-7 days, 8-14 days, and 2-8 weeks from symptom onset, respectively. Negative Percent Agreement (NPA) was 97.53% (95% CI: 93.80% to 99.32%). There was no cross-reactivity with the SARS-CoV-2 IgG antibody. Hemoglobin (200 mg/dL), bilirubin (2 mg/dL), triglyceride (250 mg/dL) and EDTA (10 mM) showed no significant interfering effect on this assay. In conclusion, an anti-SARS-CoV-2 IgM antibody assay with high sensitivity and specificity has been developed. With the high throughput, this assay will speed up the anti-SARS-CoV-2 IgM testing.

Kinetics of plasma cfDNA predicts clinical response in non-small cell lung cancer patients.

Tyrosine kinase inhibitors (TKIs), VEGF/VEGF receptor inhibitors (VEGFIs) and immune checkpoint inhibitors (ICIs) have revolutionized the treatment of advanced cancers including non-small-cell lung cancer (NSCLC). This study aims to evaluate the utility of plasma cell-free DNA (cfDNA) as a prognostic biomarker and efficacy predictor of chemotherapy (CT) with or without these precision therapies in NSCLC patients. Peripheral cfDNA levels in 154 NSCLC patients were quantified before and after the first target cycle of chemotherapy. The correlations of cfDNA with tumor burden, clinical characteristics, progression-free survival (PFS)/disease-free survival (DFS), objective response ratio (ORR), and therapy regimens were analyzed respectively. Baseline cfDNA, but not post-chemotherapeutic cfDNA, positively correlates with tumor burden. Notably, cfDNA kinetics (cfDNA Ratio, the ratio of post-chemotherapeutic cfDNA to baseline cfDNA) well distinguished responsive individuals (CR/PR) from the non-responsive (PD/SD). Additionally, cfDNA Ratio was found negatively correlated with PFS in lung adenocarcinoma (LUAD), but not lung squamous-cell carcinoma (LUSC) which may be due to a limited number of LUSC patients in this cohort. LUAD patients with low cfDNA Ratio have prolonged PFS and improved ORR, compared to those with high cfDNA Ratio. When stratified by therapy regimen, the predictive value of cfDNA Ratio is significant in patients with chemotherapy plus VEGFIs, while more patients need be included to validate the value of cfDNA Ratio in other regimens. Thus, the kinetics of plasma cfDNA during chemotherapy may function as a prognostic biomarker and efficacy predictor for NSCLC patients.

Saliva as a testing specimen with or without pooling for SARS-CoV-2 detection by multiplex RT-PCR test.

BACKGROUND: Sensitive and high throughput molecular detection assays are essential during the coronavirus disease 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The vast majority of the SARS-CoV-2 molecular assays use nasopharyngeal swab (NPS) or oropharyngeal swab (OPS) specimens collected from suspected individuals. However, using NPS or OPS as specimens has apparent drawbacks, e.g. the collection procedures for NPS or OPS specimens can be uncomfortable to some people and may cause sneezing and coughing which in turn generate droplets and/or aerosol particles that are of risk to healthcare workers, requiring heavy use of personal protective equipment. There have been recent studies indicating that self-collected saliva specimens can be used for molecular detection of SARS-CoV-2 and provides more comfort and ease of use for the patients. Here we report the performance of QuantiVirus™ SARS-CoV-2 test using saliva as the testing specimens with or without pooling. METHODS: Development and validation studies were conducted following FDA-EUA and molecular assay validation guidelines. Using SeraCare Accuplex SARS-CoV-2 reference panel, the limit of detection (LOD) and clinical performance studies were performed with the QuantiVirus™ SARS-CoV-2 test. For clinical evaluation, 85 known positive and 90 known negative clinical NPS samples were tested. Additionally, twenty paired NPS and saliva samples collected from recovering COVID-19 patients were tested and the results were further compared to that of the Abbott m2000 SARS-CoV-2 PCR assay. Results of community collected 389 saliva samples for COVID-19 screening by QuantiVirus™ SARS-CoV-2 test were also obtained and analyzed. Additionally, testing of pooled saliva samples was evaluated. RESULTS: The LOD for the QuantiVirus™ SARS-CoV-2 test was confirmed to be 100-200 copies/mL. The clinical performance studies using contrived saliva samples indicated that the positive percentage agreement (PPA) of the QuantiVirus™ SARS-CoV-2 test is 100% at 1xLOD, 1.5xLOD and 2.5xLOD. No cross-reactivity was observed for the QuantiVirus™ SARS-CoV-2 test with common respiratory pathogens. Testing of clinical samples showed a positive percentage agreement (PPA) of 100% (95% CI: 94.6% to 100%) and a negative percentage agreement (NPA) of 98.9% (95% CI: 93.1% to 99.9%). QuantiVirus™ SARS CoV-2 test had 80% concordance rate and no significant difference (p = 0.13) between paired saliva and NPS specimens by Wilcoxon matched pairs signed rank test. Positive test rate was 1.79% for 389 saliva specimens collected from local communities for COVID-19 screening. Preliminary data showed that saliva sample pooling up to 6 samples (1:6 pooling) for SARS-CoV-2 detection is feasible (sensitivity 94.8% and specificity 100%). CONCLUSION: The studies demonstrated that the QuantiVirus™ SARS-CoV-2 test has a LOD of 200 copies/mL in contrived saliva samples. The clinical performance of saliva-based testing is comparable to that of NPS-based testing. Pooling of saliva specimens for SARS-CoV-2 detection is feasible. Saliva based and high-throughput QuantiVirus™ SARS-CoV-2 test offers a highly desirable testing platform during the ongoing COVID-19 pandemic.

A high-throughput Anti-SARS-CoV-2 IgG testing platform for COVID-19.

BACKGROUND: Serology tests for detecting the antibodies to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can identify previous infection and help to confirm the presence of current infection. OBJECTIVE: The aim of this study was to evaluate the performances of a newly developed high throughput immunoassay for anti-SARS-CoV-2 IgG antibody detection. RESULTS: Clinical agreement studies were performed in 107 COVID-19 patient serum samples and 226 negative donor serum/plasma samples. Positive percent agreement (PPA) was 46.15 % (95 % CI: 19.22 % ∼74.87 %), 61.54 % (95 % CI: 31.58 % ∼86.14 %), and 97.53 % (95 % CI: 91.36 % ∼99.70 %) for samples collected on 0-7 days, 8-14 days, and ≥15 days from symptom onset, respectively. Negative Percent Agreement (NPA) was 98.23 % (95 % CI: 95.53 % ∼99.52 %). No cross-reactivity was observed to patient samples positive for IgG antibodies against the following pathogens: HIV, HAV, HBV, RSV, CMV, EBV, Rubella, Influenza A, and Influenza B. Hemoglobin (200 mg/dL), bilirubin (2 mg/dL) and EDTA (10 mM) showed no significant interfering effect on this assay. CONCLUSION: An anti-SARS-CoV-2 IgG antibody assay with high sensitivity and specificity has been developed. With the high throughput, this assay will speed up anti-SARS-CoV-2 IgG testing.

Surface-enhanced Raman scattering tags for rapid and homogeneous detection of circulating tumor cells in the presence of human whole blood.

A novel, homogeneous SERS-based cell detection assay was developed for rapid and direct enumeration of circulating tumor cells in the presence of whole blood. Magnetic beads and SERS tags were respectively conjugated to EpCAM and her2 antibodies for the capture and detection of approximately 50 tumor cells/mL in the presence of whole blood in less than 1 h.

The zeta potential of surface-functionalized metallic nanorod particles in aqueous solution.

Metallic nanoparticles suspended in aqueous solutions and functionalized with chemical and biological surface coatings are important elements in basic and applied nanoscience research. Many applications require an understanding of the electrokinetic or colloidal properties of such particles. We describe the results of experiments to measure the zeta potential of metallic nanorod particles in aqueous saline solutions, including the effects of pH, ionic strength, metallic composition, and surface functionalization state. Particle substrates tested include gold, silver, and palladium monometallic particles as well as gold/silver bimetallic particles. Surface functionalization conditions included 11-mercaptoundecanoic acid (MUA), mercaptoethanol (ME), and mercaptoethanesulfonic acid (MESA) self-assembled monolayers (SAMs), as well as MUA layers subsequently derivatized with proteins. For comparison, we present zeta potential data for typical charge-stabilized polystyrene particles. We compare experimental zeta potential data with theoretically predicted values for SAM-coated and bimetallic particles. The results of these studies are useful in predicting and controlling the aggregation, adhesion, and transport of functionalized metallic nanoparticles within microfluidic devices and other systems.

SERS nanoparticles: a new optical detection modality for cancer diagnosis.

Surface-enhanced Raman scattering (SERS) is an optical detection technique that offers advantages over traditional assay detection technologies, such as fluorescence and chemiluminescence. These advantages include sensitivity, high levels of multiplexing, robustness and ability to perform detection in blood and other biological matrices. Here, we report on the growing field of SERS-active nanoparticles as a novel method for detection, with special emphasis on their use in the field of oncology. We discuss examples of SERS-active nanoparticles used in an assay for PSA, BRCA1 and Her-2, along with examples of nucleic-acid detection. We present data on a novel homogeneous, single-tube, rapid assay for nucleic acid detection and show how it will benefit the oncology community.

Silica-coated, Au/Ag striped nanowires for bioanalysis.

Striped metallic nanowires (NW) have been coated with a silica shell of controllable thickness (6-150 nm), and the assay performance of coated vs uncoated NW has been compared. The silica coating does not interfere with identification of the metal striping pattern and protects Ag segments from oxidation, extending the range of assay conditions under which barcoded NW can be used. Much higher and more uniform fluorescence intensities were observed for dye-labeled ssDNA bound to SiO2-coated as compared to intensities for uncoated NW. Simultaneous, multiplexed DNA hybridization assays for three pathogen-specific target sequences on SiO2-coated NW showed good discrimination of complementary from noncomplementary targets. Application of SiO2-coated NW in discrimination of single base mismatches corresponding to a mutation of the p53 gene was also demonstrated. Finally, we have shown that thiolated probe DNA resists desorption under thermocycling conditions if attached via siloxane chemistry to SiO2-coated NW, but not if it is attached via direct adsorption to bare Au/Ag NW.

Coupling molecular beacons to barcoded metal nanowires for multiplexed, sealed chamber DNA bioassays.

We have combined molecular beacon (MB) probes with barcoded metal nanowires to enable no-wash, sealed chamber, multiplexed detection of nucleic acids. Probe design and experimental parameters important in nanowire-based MB assays are discussed. Loop regions of 24 bases and 5 base pair stem regions in the beacon probes gave optimal performance. Our results suggest that thermodynamic predictions for secondary structure stability of solution-phase MB can guide probe design for nanowire-based assays. Dengue virus-specific probes with predicted solution-phase DeltaG of folding in 500 mM buffered NaCl of approximately -4 kcal/mol performed better than those with DeltaG > -2 or < -6 kcal/mol. Buffered 300-500 mM NaCl was selected after comparison of several buffers previously reported for similar types of assays, and 200-500 mM NaCl was found to be the optimal ionic strength for the hybridization temperatures (25 and 50 degrees C) and probe designs used here. Target binding to the surface as a function of solution concentration fit a Sips isotherm with Kd = 1.7 +/- 0.3 nM. The detection limit was approximately 100 pM, limited by incomplete quenching. Single base mismatches could be discriminated from fully complementary targets. Oligonucleotide target sequences specific for human immunodeficiency, hepatitis C, and severe acute respiratory viruses were assayed simultaneously in a no-wash, sealed chamber, multiplexed experiment in which each of three probe sequences was attached to a different pattern of encoded nanowires. Finally, we demonstrated that probe-coated nanowires retain their selectivity and sensitivity in a triplexed assay after storage for over 3 months.

Multiplexed SNP genotyping using nanobarcode particle technology.

Single-nucleotide polymorphisms (SNP) are the most common form of sequence variation in the human genome. Large-scale studies demand high-throughput SNP genotyping platforms. Here we demonstrate the potential of encoded nanowires for use in a particles-based universal array for high-throughput SNP genotyping. The particles are encoded sub-micron metallic nanorods manufactured by electroplating inert metals such as gold and silver into templates and releasing the resulting striped nanoparticles. The power of this technology is that the particles are intrinsically encoded by virtue of the different reflectivity of adjacent metal stripes, enabling the generation of many thousands of unique encoded substrates. Using SNP found within the cytochrome P450 gene family, and a universal short oligonucleotide ligation strategy, we have demonstrated the simultaneous genotyping of 15 SNP; a format requiring discrimination of 30 encoded nanowires (one per allele). To demonstrate applicability to real-world applications, 160 genotypes were determined from multiplex PCR products from 20 genomic DNA samples.

Use of nanobarcodes particles in bioassays.

We have developed striped metal nanoparticles, Nanobarcodes particles, which can act as encoded substrates in multiplexed assays. These particles are metallic, encodeable, machine-readable, durable, submicron-sized tags. The power of this technology is that the particles are intrinsically encoded by virtue of the difference in reflectivity of adjacent metal stripes. This chapter describes protocols for the attachment of biological molecules, and the subsequent use of the Nanobarcodes particles in bioassays.

Encoded metal nanoparticle-based molecular beacons for multiplexed detection of DNA.

In this paper we describe a molecular beacon format assay in which encoded nanowire particles are used to achieve multiplexing. We demonstrate this principle with the detection of five viral pathogens; Hepatitis A virus, Hepatitis C virus, West Nile Virus, Human Immune Deficiency virus and Severe Acute Respiratory Syndrome virus. Oligonucleotides are designed complementary to a target sequence of interest containing a 3' universal fluorescence dye. A 5' thiol causes the oligonucleotides to self-assemble onto the metal nanowire. The single-stranded oligonucleotide contains a self-complementary hairpin stem sequence of 10 bases that forces the 3' fluorophore to come into contact with the metallic nanowire surface, thereby quenching the fluorescence. Upon addition of target DNA, there is hybridization with the complementary oligonucleotides. The resulting DNA hybrid is rigid, unfolds the hairpin structure, and causes the fluorophore to be moved away from the surface such that it is no longer quenched. By using differently encoded nanowires, each conjugated with a different oligonucleotide sequence, multiplexed DNA assays are possible using a single fluorophore, from a multiplexed RT-PCR reaction.

Multiplexed SNP genotyping using the Qbead system: a quantum dot-encoded microsphere-based assay.

We have developed a new method using the Qbead system for high-throughput genotyping of single nucleotide polymorphisms (SNPs). The Qbead system employs fluorescent Qdot semiconductor nanocrystals, also known as quantum dots, to encode microspheres that subsequently can be used as a platform for multiplexed assays. By combining mixtures of quantum dots with distinct emission wavelengths and intensities, unique spectral 'barcodes' are created that enable the high levels of multiplexing required for complex genetic analyses. Here, we applied the Qbead system to SNP genotyping by encoding microspheres conjugated to allele-specific oligonucleotides. After hybridization of oligonucleotides to amplicons produced by multiplexed PCR of genomic DNA, individual microspheres are analyzed by flow cytometry and each SNP is distinguished by its unique spectral barcode. Using 10 model SNPs, we validated the Qbead system as an accurate and reliable technique for multiplexed SNP genotyping. By modifying the types of probes conjugated to microspheres, the Qbead system can easily be adapted to other assay chemistries for SNP genotyping as well as to other applications such as analysis of gene expression and protein-protein interactions. With its capability for high-throughput automation, the Qbead system has the potential to be a robust and cost-effective platform for a number of applications.