Combining Bioorthogonal Chemistry with Fluorescent Silica Nanoparticles for the Ultrasensitive Detection of the HIV-1 p24 Antigen.

Early detection and viral concentration monitoring of human immunodeficiency virus in resource-poor settings are important to control disease spread and reduce mortality. Nucleic acid amplification tests are expensive for low-resource settings. Lateral flow antibody tests are not sensitive if testing is performed within 7-10 days, and these tests are not quantitative. We describe a signal enhancement technique based on fluorescent silica nanoparticles and bioorthogonal chemistries for the femtomolar detection of the HIV-1 p24 antigen. We developed a magnetic bead-based assay, wherein we used fluorescent-dye-encapsulated silica nanoparticles as reporters. The number of reporters was increased by using bioorthogonal chemistry to provide signal enhancement. The limit and range of detection of the sandwich immunoassay using alternating multiple layers for p24 in human serum were found to be 46 fg/mL (1.84 fM) and 46 fg/mL to 10 ng/mL, respectively. This simple assay was 217-fold higher in sensitivity compared to that of commercial enzyme-linked immunoassays (limit of detection of 10 pg/mL).

Palladium encapsulated mesoporous silica nanoparticles for the rapid detection of analytes.

We designed a simple, inexpensive, and user-friendly assay using mesoporous silica nanoparticles to detect analytes. Highly stable and uniform palladium nanoparticles covered with mesoporous silica (Pd@mSiO2) were generated and characterized extensively using physical methods. Human Serum Albumin (HSA) protein or ssDNA specific to the HIV gag region was capped onto the Pd@mSiO2 electrostatically. This "cap" prevented the Pd(0) inside the mesoporous silica nanoparticles from catalyzing the conversion of non-fluorescent molecules to fluorescent molecules. In the presence of target anti-HSA antibodies or complementary sequence (HIV gag), HSA protein or DNA cap dissociated from the surface of Pd@mSiO2-NH2 through the specific antigen-antibody reaction or DNA hybridization, allowing Pd(0) to convert the non-fluorescent molecules to fluorescent molecules. The limit and range of detection of anti-HSA antibodies were 3.8 nM and 3.8 nM to 133.3 nM, respectively. The limit and range of detection of HIV gag were 1.6 nM and 1.6 nM to 15 nM, respectively. This simple, inexpensive, "add sample and measure" diagnostic assay could potentially be incorporated into point of care diagnostics for low-resource settings.

Inflammatory bowel disease biomarkers.

Inflammatory bowel disease (IBD) is characterized as chronic inflammation in the gastrointestinal tract, which includes two main subtypes, Crohn's disease and ulcerative colitis. Endoscopy combined with biopsy is the most effective way to establish IBD diagnosis and disease management. Imaging techniques have also been developed to monitor IBD. Although effective, the methods are expensive and invasive, which leads to pain and discomfort. Alternative noninvasive biomarkers are being explored as tools for IBD prognosis and disease management. This review focuses on novel biomarkers that have emerged in recent years. These serological biomarkers and microRNAs could potentially be used for disease management in IBD, thereby decreasing patient discomfort and morbidity.

Toward Point-of-Care Diagnostics to Monitor MMP-9 and TNF-α Levels in Inflammatory Bowel Disease.

We have investigated the association of matrix metallopeptidase 9 (MMP-9) and tumor necrosis factor α (TNF-α) levels with colitis severity using an established IL10-/- mouse model, which reflects the severity of inflammation in humans with inflammatory bowel disease (IBD). We found that MMP-9 and TNF-α correlated with colitis severity. In parallel, we developed assays to detect fecal MMP-9 and serum TNF-α using "cap and release" mesoporous silica nanoparticles (MSNs). MMP-9 peptide substrates as "caps" were attached to dye-loaded MSNs. The introduction of MMP-9 resulted in substrate cleavage and subsequent dye release, which was rapidly detected using a fluorometer. For TNF-α, an anti-TNF antibody was used as the "cap". The introduction of TNF-α antigen leads to the release of the dyes because the antigen binds more strongly to the antibody cap. The MSN-based assays can detect MMP-9 and TNF-α effectively, although signal amplification is required to meet clinical sensitivity.

ASSURED-SQVM diagnostics for COVID-19: addressing the why, when, where, who, what and how of testing.

Introduction: SARS-CoV-2, the new coronavirus that originated in 2019, continues to impact every aspect of society in a profound manner. Testing will remain an important tool to mitigate the effects of this pandemic as early and accurate diagnosis can lead to appropriate countermeasures to reduce mortality and morbidity. However, testing isn't a simple yes/no answer as the target and host are complex, the virus is a moving target, there is a plethora of tests that identify different parts of the virus and have their own limits and range of detection, and when prevalence is low, false positives and negatives can be very high.Areas covered: This article covers all the major questions related to COVID-19 diagnostics, the why, when, where, who, what and how of testing, the different types of tests, interpretation of results and the ideal ASSURED-SQVM diagnostic. A comprehensive literature review using all the publicly available databases and government websites and reports was performed.Expert opinion: Diagnostics that meet the 'ASSURED-SQVM' (Affordable, Selective and Sensitive, User-friendly, Rapid and Robust, Equipment-free, Deliverable to end-users and additionally, allows for Self-testing, Quantifiable, detects if pathogens are Viable and can detect Multiple pathogens) would make a major impact in our fight against the current pandemic. While a significant majority of researchers focus on developing novel diagnostics that are highly selective and sensitive, it is the opinion of these authors that other aspects of the ASSURED-SQVM principles also be considered early in the development process for widespread use.

Point-of-Care Monitoring of Colitis Using Intestinal Alkaline Phosphatase in Inflammatory Bowel Disease.

Intestinal Alkaline Phosphatase (IAP) was investigated as a potential biomarker to monitor colitis in a mouse model of Inflammatory Bowel Disease (IBD). We developed a Point-Of-Care (POC) assay to detect IAP with a glucose meter in 15 min. We synthesized a paracetamol-bearing compound specifically cleaved by IAP to release paracetamol, which can be detected with a personal glucometer. Interleukin 10 deficient (IL 10-/-) mouse model samples were used to compare the IAP level in mice with mild or severe colitis. The results showed that fecal IAP level was significantly lower in each mouse sample with severe colitis than with mild colitis. Mice treated with anti-Tumor Necrosis Factor-alpha (anti-TNF-α) to decrease inflammation exhibited a much higher level of IAP than those without treatment (IAP levels from anti-TNF-α treated vs nontreated = 2.80 U vs 0.11 U, P < 0.0001). Taken together, IAP can be considered as a potential biomarker to monitor colitis, and a rapid, user-friendly POC glucometer-based assay can be potentially used to monitor colitis levels and inflammation flareups in IBD.

Rapid, user-friendly, and inexpensive detection of azidothymidine.

Strict adherence to highly active antiretroviral therapy (HAART) is very important to improve the quality of life for HIV-positive patients to reduce new infections and determine treatment success. Azidothymidine (AZT) is an antiretroviral drug commonly used in HAART treatment. In this research, an "add, mix, and measure" assay was developed to detect AZT within minutes. Three different probes designed to release fluorophores when samples containing AZT are added were synthesized and characterized. The limit of detection to AZT in simulated urine samples was determined to be 4 µM in 5 min for one of the probes. This simple and rapid point-of-care test could potentially be used by clinicians and health care workers to monitor the presence of AZT in low resource settings.

Fluorescent sialic derivatives for the specific detection of influenza viruses.

Early and accurate diagnosis of influenza viruses can decrease its harmful impact. Here, we have synthesized fluorescent sialic acid derivatives that are cleaved by influenza neuraminidases (NAs) and not by Streptococcus pneumoniae that also inhabits the human olfactory. We have also attempted to develop assays that could differentiate between influenza virus and S. pneumoniae by taking advantage of the structural differences between NAs from these pathogens.

Detection of Enzymes, Viruses, and Bacteria Using Glucose Meters.

We have developed innovative assays that can detect enzymes rapidly. Paracetamol- or catechol-bearing compounds, when exposed to their respective enzymes, released paracetamol or catechol, which can be detected using a standard glucose meter. This approach was used to detect a number of diverse analytes that include enzymes such as ß-galactosidase and α-mannosidase and pathogens such as influenza viruses, Streptococcus pneumoniae, and E. coli rapidly. The limit of detection for all analytes was extremely low and clinically relevant for influenza viruses. We also demonstrate that glucose oxidase or glucose dehydrogenase is not required because the paracetamol gets oxidized directly on the electrode surface. This indicates that test strips without glucose oxidase or dehydrogenase can be used, and we can detect analytes in the presence of high levels of background glucose. We demonstrate this unique nature of the assay to detect paracetamol in simulated urine and sheep blood without background interference of intrinsic glucose, indicating that glucose meters can be used to detect nonglucose analytes without background glucose interference.

Multivalent oleanolic acid human serum albumin conjugate as nonglycosylated neomucin for influenza virus capture and entry inhibition.

We report the synthesis of multivalent oleanolic acid (OA) protein conjugates as nonglycosylated neomucin mimic for the capture and entry inhibition of influenza viruses. Oleanolic acid derivatives bearing an amine-terminated linker were synthesized by esterification of carboxylic acid and further grafted onto the human serum albumin (HSA) via diethyl squarate method. The binding of hemagglutinin (HA) on the virion surface to the synthetic neomucin was evaluated by hemagglutination inhibition assay. The influenza virus capture ability of the PEGylated OA-HSA conjugate was further investigated by Dynamic Light Scattering (DLS), virus capture assay and Isothermal Titration Calorimeter (ITC) techniques. The pronounced agglutination of viral particles, the high capture efficiency and affinity constant indicate that this neoprotein is comparable to natural glycosylated mucin, suggesting that this material could potentially be used as anti-infective barriers to prevent virus from invading host cells. The study also rationalizes the feasibility of antiviral drug development based on OA or other antiviral small molecules conjugated protein strategies.

Highly specific and rapid glycan based amperometric detection of influenza viruses.

Rapid and precise detection of influenza viruses in a point of care setting is critical for applying appropriate countermeasures. Current methods such as nucleic acid or antibody based techniques are expensive or suffer from low sensitivity, respectively. We have developed an assay that uses glucose test strips and a handheld potentiostat to detect the influenza virus with high specificity. Influenza surface glycoprotein neuraminidase (NA), but not bacterial NA, cleaved galactose bearing substrates, 4,7di-OMe N-acetylneuraminic acid attached to the 3 or 6 position of galactose, to release galactose. In contrast, viral and bacterial NA cleaved the natural substrate, N-acetylneuraminic acid attached to the 3 or 6 position of galactose. The released galactose was detected amperometrically using a handheld potentiostat and dehydrogenase bearing glucose test strips. The specificity for influenza was confirmed using influenza strains and different respiratory pathogens that include Streptococcus pneumoniae and Haemophilus influenzae; bacteria do not cleave these molecules. The assay was also used to detect co-infections caused by influenza and bacterial NA. Viral drug susceptibility and testing with human clinical samples was successful in 15 minutes, indicating that this assay could be used to rapidly detect influenza viruses at primary care or resource poor settings using ubiquitous glucose meters.

Multivalent S-sialoside protein conjugates block influenza hemagglutinin and neuraminidase.

A new class of S-sialoside Human Serum Albumin (HSA) and Bovine Serum Albumin (BSA) conjugates were prepared to enhance the binding affinity to hemagglutinin (HA) and neuraminidase (NA). The valency of glycoconjugates was controlled by the reaction ratio of the S-sialoside monomer and protein. Hemagglutination inhibition assay showed that these synthetic glycoproteins have higher affinity to HA than the small clusters of sialosides with lower valency, due to multivalent effect and optimized three dimensional presentation of sialosides on the protein platform. The results of fluorescent NA inhibition assay showed that some of the conjugates have moderate NA inhibitory activity, in comparison to the monomer and low valent conjugates with weak or none inhibitory activity. These synthetic sialylated proteins were not cytotoxic with concentrations up to 100 µM, since the sialylation did not change the secondary structure of protein. This new kind of conjugates can be used as lead compounds for antiviral drug design and the construction of pseudo sialoside-protein conjugates library to investigate the carbohydrate-HA/NA recognition process and a platform for the influenza virus capturing.

Toward the Development of the Next Generation of a Rapid Diagnostic Test: Synthesis of Glycophosphatidylinositol (GPI) Analogues of Plasmodium falciparum and Immunological Characterization.

A large number of proteins in malaria parasites are anchored using glycophosphatidylinositols (GPIs) with lipid tails. These GPIs are structurally distinct from human GPIs. Plasmodium falciparum GPIs have been considered as potential vaccine candidates because these molecules are involved in inducing inflammatory responses in human hosts, and natural anti-GPI antibody responses have been shown to be associated with protection against severe disease. GPIs can also be considered as targets for rapid diagnostic tests. Because isolation of native GPIs in large quantities is challenging, development of synthetic GPI molecules can facilitate further exploration of GPI molecules for diagnostics. Here, we report synthesis and immunological characterization of a panel of malaria-specific GPI analogues. A total of three GPI analogues were chemically synthesized and conjugated to a carrier protein to immunize and generate antibodies in rabbits. The rabbit immune sera showed reactivity with synthetic GPIs and native GPIs extracted from P. falciparum parasite, as determined by Luminex and ELISA methods.

Synthesis and Evaluation of Biotinylated Bivalent HistoBlood Group Antigens for Capturing Human Noroviruses.

A panel of biotinylated bivalent H-type glycans that have been reported as binding ligands for human noroviruses were synthesized using a modular synthetic strategy. These glycoconjugates were attached to streptavidin-coated magnetic beads and used to recover human norovirus from fecal samples using a magnetic bead-based assay. The biotinylated bivalent glycans synthesized for this study exhibited similar or better capturing ability when compared to commercial biotinylated glycopolymers.

Polyvalent effect enhances diglycosidic antiplasmodial activity.

An efficient and facile total synthesis of diglycoside Matayoside D isolated from the root bark of Matayba guianensis with antiplasmodial activity have been accomplished in 11 steps with 5% overall yields starting from commercially available glucose and rhamnose. Furthermore, a class of the diglycosidic derivatives with different lengths of the linker and valences were also prepared and evaluated for their antiplasmodial activities against chloroquine-susceptible (3D7) and chloroquine-resistant (W2) strains of Plasmodium falciparum. Low valent and short linker attached diglycoside show no enhancement of the antiplasmodial activity while polyvalent conjugates showed enhanced antiplasmodial activity with IC50 value at least 20 fold better than that of the corresponding diglycosidic monomer. The polyvalent diglycoside were non-cytotoxic against normal mammalian cells under 50,000 µg/L.

Neuraminidase Resistant Sialosides for the Detection of Influenza Viruses.

We report the synthesis of influenza virus neuraminidase (NA) resistant sialosides that include different glycoside linkages (C-, S-, and triazole). These unnatural sialosides were printed onto glass slides to generate a small focused microarray. We evaluated the binding affinity of multiple lectins and compared the stability of these sialosides with O-linked sialosides toward influenza virus neuraminidase and intact virus. We demonstrated the ability of these molecules to capture eight different strains of influenza virus at ambient temperature without the addition of NA inhibitors. The glycans capture extremely low, clinically relevant concentrations of viruses and each strain gives rise to a specific "fingerprint" binding pattern, which could potentially be used in rapid diagnostic tests.

Indirect Detection of Glycosidases Using Amperometry.

Glycosidases are essential enzymes that cleave glycoside bonds. The presence of glycosidases have been widely used to detect pathogens, label cells/tissues, and report specific diseases. We have developed a rapid electrochemical assay to detect glycosidases. Exposure of electrochemically inactive substrates to glycosidases releases glucose, which can be measured easily using an electrochemical cell. Five different glycosidases were detected rapidly within 1 h using disposable electrodes. This assay could readily be incorporated into repurposed glucose meters to rapidly detect glycosidases, which in turn could be useful to report the presence of a pathogen or illness.

Electrochemical assay to detect influenza viruses and measure drug susceptibility.

An electrochemical assay has been designed to rapidly diagnose influenza viruses. Exposure of a glucose-bearing substrate to influenza viruses or its enzyme, neuraminidase (NA), releases glucose, which was detected amperometrically. Two methods were used to detect released glucose. First, we used a standard glucose blood meter to detect two viral NAs and three influenza strains. We also demonstrated drug susceptibility of two antivirals, Zanamivir and Oseltamivir, using the assay. Finally, we used disposable test strips to detect nineteen H1N1 and H3N2 influenza strains using this assay in one hour. The limit and range of detection of this first generation assay is 10(2) and 10(2)-10(8) plaque forming units (pfu), respectively. Current user-friendly glucose meters can be repurposed to detect influenza viruses.

Glycan based detection and drug susceptibility of influenza virus.

We have developed a panel of synthetic glycans as receptor mimics for the specific capture of influenza viruses. The glycans were printed onto commercial glass slides using a free amine at the end of a spacer to generate a small focused microarray. The microarray was evaluated for its ability to capture three different strains of influenza A virus, two H1N1, A/Brisbane/59/2007 and A/Solomon Islands/3/2006 and one H3N2, A/Aichi/2/1968. We observed an excellent detection ability with some compounds exhibiting clinically relevant (10(1) plaque forming units) limit of detection. We also tested the drug susceptibility of current antivirals, Zanamivir and Ostelamivir using this microarray and could determine antiviral resistance for these strains.

Bifunctional thiosialosides inhibit influenza virus.

We have synthesized a panel of bivalent S-sialoside analogues, with modifications at the 4 position, as inhibitors of influenza virus. These first generation compounds show IC50 values ranging from low micromolar to high nanomolar in enzyme inhibition and plaque reduction assays with two intact viruses, Influenza H1N1 (A/California/07/2009) and H3N2 (A/Hongkong/8/68).