Sensitive Blood-Based Detection of HIV-1 and Mycobacterium tuberculosis Peptides for Disease Diagnosis by Immuno-Affinity Liquid Chromatography-Tandem Mass Spectrometry: A Method Development and Proof-of-Concept Study.

BACKGROUND: Novel approaches that allow early diagnosis and treatment monitoring of both human immunodeficiency virus-1 (HIV-1) and tuberculosis disease (TB) are essential to improve patient outcomes. METHODS: We developed and validated an immuno-affinity liquid chromatography-tandem mass spectrometry (ILM) assay that simultaneously quantifies single peptides derived from HIV-1 p24 and Mycobacterium tuberculosis (Mtb) 10-kDa culture filtrate protein (CFP10) in trypsin-digested serum derived from cryopreserved serum archives of cohorts of adults and children with/without HIV and TB. RESULTS: ILM p24 and CFP10 results demonstrated good intra-laboratory precision and accuracy, with recovery values of 96.7% to 104.6% and 88.2% to 111.0%, total within-laboratory precision (CV) values of 5.68% to 13.25% and 10.36% to 14.92%, and good linearity (r2 > 0.99) from 1.0 to 256.0 pmol/L and 0.016 to 16.000 pmol/L, respectively. In cohorts of adults (n = 34) and children (n = 17) with HIV and/or TB, ILM detected p24 and CFP10 demonstrated 85.7% to 88.9% and 88.9% to 100.0% diagnostic sensitivity for HIV-1 and TB, with 100% specificity for both, and detected HIV-1 infection earlier than 3 commercial p24 antigen/antibody immunoassays. Finally, p24 and CFP10 values measured in longitudinal serum samples from children with HIV-1 and TB distinguished individuals who responded to TB treatment from those who failed to respond or were untreated, and who developed TB immune reconstitution inflammatory syndrome. CONCLUSIONS: Simultaneous ILM evaluation of p24 and CFP10 results may allow for early TB and HIV detection and provide valuable information on treatment response to facilitate integration of TB and HIV diagnosis and management.

Monocrystalline Labeling Enables Stable Plasmonic Enhancement for Isolation-Free Extracellular Vesicle Analysis.

Sensitive detection of extracellular vesicles (EVs) as emerging biomarkers has shown great promises for disease diagnosis. Plasmonic metal nanostructures conjugated with molecules that bind specific biomarker targets are widely used for EVs sensing but involve tradeoffs between particle-size-dependent signal intensity and conjugation efficiency. One solution to this problem would be to induce nucleation on nanoparticles that have successfully bound a target biomarker to permit in situ nanoparticle growth for signal amplification, but approaches that are evaluated to date require harsh conditions or lack nucleation specificity, prohibiting their effective use with most biological specimens. This study describes a one-step in situ strategy to induce monocrystalline copper shell growth on gold nanorod probes without decreasing signal by disrupting probe-target interactions or lipid bilayer integrity to enable EV biomarker detections. This approach increases the detected nanoparticle signal about two orders of magnitude after a 10 min copper nanoshell growth reaction. This has significant implications for improved disease detection, as indicated by the ability of a novel immunoassay using this approach to detect low abundance EVs carrying a pathogen-derived biomarker, after their direct capture from serum, to facilitate the diagnosis of tuberculosis cases in a diagnostically challenging pediatric cohort.

Diagnosis of paediatric tuberculosis by optically detecting two virulence factors on extracellular vesicles in blood samples.

Sensitive and specific blood-based assays for the detection of pulmonary and extrapulmonary tuberculosis would reduce mortality associated with missed diagnoses, particularly in children. Here we report a nanoparticle-enhanced immunoassay read by dark-field microscopy that detects two Mycobacterium tuberculosis virulence factors (the glycolipid lipoarabinomannan and its carrier protein) on the surface of circulating extracellular vesicles. In a cohort study of 147 hospitalized and severely immunosuppressed children living with HIV, the assay detected 58 of the 78 (74%) cases of paediatric tuberculosis, 48 of the 66 (73%) cases that were missed by microbiological assays, and 8 out of 10 (80%) cases undiagnosed during the study. It also distinguished tuberculosis from latent-tuberculosis infections in non-human primates. We adapted the assay to make it portable and operable by a smartphone. With further development, the assay may facilitate the detection of tuberculosis at the point of care, particularly in resource-limited settings.

SARS-CoV-2 Epitopes following Infection and Vaccination Overlap Known Neutralizing Antibody Sites.

Identification of epitopes targeted following virus infection or vaccination can guide vaccine design and development of therapeutic interventions targeting functional sites, but can be laborious. Herein, we employed peptide microarrays to map linear peptide epitopes (LPEs) recognized following SARS-CoV-2 infection and vaccination. LPEs detected by nonhuman primate (NHP) and patient IgMs after SARS-CoV-2 infection extensively overlapped, localized to functionally important virus regions, and aligned with reported neutralizing antibody binding sites. Similar LPE overlap occurred after infection and vaccination, with LPE clusters specific to each stimulus, where strong and conserved LPEs mapping to sites known or likely to inhibit spike protein function. Vaccine-specific LPEs tended to map to sites known or likely to be affected by structural changes induced by the proline substitutions in the mRNA vaccine's S protein. Mapping LPEs to regions of known functional importance in this manner may accelerate vaccine evaluation and discovery of targets for site-specific therapeutic interventions.

CRISPR detection of circulating cell-free Mycobacterium tuberculosis DNA in adults and children, including children with HIV: a molecular diagnostics study.

BACKGROUND: Tuberculosis remains a leading cause of global mortality, especially for adults and children living with HIV (CLHIV) underdiagnosed by sputum-based assays. Non-sputum-based assays are needed to improve tuberculosis diagnosis and tuberculosis treatment monitoring. Our aim in this study was to determine whether ultrasensitive detection of Mycobacterium tuberculosis cell-free DNA (Mtb-cfDNA) in blood can diagnose tuberculosis and evaluate tuberculosis treatment responses. METHODS: In this molecular diagnostics study we analysed archived serum from two patient populations evaluated for tuberculosis in Eswatini and Kenya to detect Mtb-cfDNA, analysing serum from all individuals who had both sufficient serum volumes and clear diagnostic results. An optimised CRISPR-mediated tuberculosis (CRISPR-TB) assay was used to detect Mtb-cfDNA in serum at enrolment from adults and children with presumptive tuberculosis and their asymptomatic household contacts, and at enrolment and during tuberculosis treatment from a cohort of symptomatic CLHIV at high risk for tuberculosis, who provided longitudinal serum at enrolment and during tuberculosis treatment. FINDINGS: CRISPR-TB identified microbiologically and clinically confirmed tuberculosis cases in the predominantly HIV-negative Eswatini adult cohort with 96% sensitivity (27 [96%] of 28, 95% CI 80-100) and 94% specificity (16 [94%] of 17, 71-100), and with 83% sensitivity (5 [83%] of 6, 36-100) and 95% specificity (21 [95%] of 22, 77-100) in the paediatric cohort, including all six cases of extrapulmonary tuberculosis. In the Kenyan CLHIV cohort, CRISPR-TB detected all (13 [100%] of 13, 75-100) confirmed tuberculosis cases and 85% (39 [85%] of 46, 71-94) of unconfirmed tuberculosis cases diagnosed by non-microbiological clinical findings. CLHIV who were CRISPR-TB positive at enrolment had a 2·4-times higher risk of mortality by 6 months after enrolment. Mtb-cfDNA signal decreased after tuberculosis treatment initiation, with near or complete Mtb-cfDNA clearance by 6 months after tuberculosis treatment initiation. INTERPRETATION: CRISPR-mediated detection of circulating Mtb-cfDNA shows promise to increase the identification of paediatric tuberculosis and HIV-associated tuberculosis, and potential for early diagnosis and rapid monitoring of tuberculosis treatment responses. FUNDING: US Department of Defense, National Institute of Child Health and Human Development, National Institute of Allergy and Infectious Diseases, University of Washington Center for AIDS Research, and the Weatherhead Presidential Endowment fund.

Nanopore-based disease diagnosis using pathogen-derived tryptic peptides from serum.

Nanopore sensors have shown great utility in nucleic acid detection and sequencing approaches. Recent studies also indicate that current signatures produced by peptide-nanopore interactions can distinguish high purity peptide mixtures, but the utility of nanopore sensors in clinical applications still needs to be explored due to the inherent complexity of clinical specimens. To fill this gap between research and clinical nanopore applications, we describe a methodology to select peptide biomarkers suitable for use in an immunoprecipitation-coupled nanopore (IP-NP) assay, based on their pathogen specificity, antigenicity, charge, water solubility and ability to produce a characteristic nanopore interaction signature. Using tuberculosis as a proof-of-principle example in a disease that can be challenging to diagnose, we demonstrate that a peptide identified by this approach produced high-affinity antibodies and yielded a characteristic peptide signature that was detectable over a broad linear range, to detect and quantify a pathogen-derived peptide from digested human serum samples with high sensitivity and specificity. This nanopore signal distinguished serum from a TB case, non-disease controls, and from a TB-case after extended anti-TB treatment. We believe this assay approach should be readily adaptable to other infectious and chronic diseases that can be diagnosed by peptide biomarkers.

Reversing Bacterial Resistance to Gold Nanoparticles by Size Modulation.

One major frustration in developing antibiotics is that bacteria can quickly develop resistance that would require an entirely new cycle of research and clinical testing to overcome. Although plenty of bactericidal nanomaterials have been developed against increasingly severe superbugs, few reports have studied the resistance to these nanomaterials. Herein, we show that antibacterial 4,6-diamino-2-pyrimidine thiol (DAPT)-capped gold nanoparticles (AuDAPTs) can induce a 16-fold increased minimum inhibitory concentration (MIC) of E. coli only after very long term exposure (183 days), without developing cross-resistance to commercialized antibiotics. Strikingly, we recovered the bactericidal activities of AuDAPTs to the resistant strain by tuning the sizes of AuDAPTs without employing new chemicals. Such slow, easy-to-handle resistance induced by AuDAPTs is unprecedented compared to traditional antibiotics or other nanomaterials. In addition to the novel antibacterial activities and biocompatibilities, our approach will accelerate the development of gold nanomaterial-based therapeutics against multi-drug-resistant (MDR) bacterial infections.

The Integrin Binding Peptide, ATN-161, as a Novel Therapy for SARS-CoV-2 Infection.

Many efforts to design and screen therapeutics for the current severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) pandemic have focused on inhibiting viral host cell entry by disrupting angiotensin-converting enzyme-2 (ACE2) binding with the SARS-CoV-2 spike protein. This work focuses on the potential to inhibit SARS-CoV-2 entry through a hypothesized α5ß1 integrin-based mechanism and indicates that inhibiting the spike protein interaction with α5ß1 integrin (+/- ACE2) and the interaction between α5ß1 integrin and ACE2 using a novel molecule (ATN-161) represents a promising approach to treat coronavirus disease-19.

Nanoplasmonic Sensor Approaches for Sensitive Detection of Disease-Associated Exosomes.

Exosomes are abundantly secreted by most cells that carry membrane and cytosolic factors that can reflect the physiologic state of their source cells and thus have strong potential to serve as biomarkers for early diagnosis, disease staging, and treatment monitoring. However, traditional diagnostic or prognostic applications that might use exosomes are hindered by the lack of rapid and sensitive assays that can exploit their biological information. An array of assay approaches have been developed to address this deficit, including those that integrate immunoassays with nanoplasmonic sensors to measure changes in optical refractive indexes in response to the binding of low concentrations of their targeted molecules. These sensors take advantage of enhanced and tunable interactions between the electron clouds of nanoplasmonic particles and structures and incident electromagnetic radiation to enable isolation-free and ultrasensitive quantification of disease-associated exosome biomarkers present in complex biological samples. These unique advantages make nanoplasmonic sensing one of the most competitive approaches available for clinical applications and point-of-care tests that evaluate exosome-based biomarkers. This review will briefly summarize the origin and clinical utility of exosomes and the limitations of current isolation and analysis approaches before reviewing the specific advantages and limitations of nanoplasmonic sensing devices and indicating what additional developments are necessary to allow the translation of these approaches into clinical applications.

Alginate Hydrogel-Embedded Capillary Sensor for Quantitative Immunoassay with Naked Eye.

We have developed an alginate hydrogel-embedded capillary sensor (AHCS) for naked eye-based quantification of immunoassay. Alkaline phosphatase (ALP) can modulate gel-sol transformation to increase the permeability of Cu2+-cross-linked alginate hydrogel film in the AHCS, followed by solution exchange into the capillary. Through measuring the length of the liquid phase of the microfluidics in the capillary at a given time, the concentration of the ALP could be quantified with the naked eye. Since ALP is widely applied as a signal reporter for immunoassays, the AHCS could easily accommodate conventional immune sensing platforms. We justify the practicality of AHCS with hepatitis B virus surface antigen (HBsAg) in serum samples and got comparable results with commercialized immunoassay. This AHCS is easy to make and use, effective in cost, and robust in quantification with the naked eye, showing great promise for next generation point-of-care testing.

The Integrin Binding Peptide, ATN-161, as a Novel Therapy for SARS-CoV-2 Infection.

Many efforts to design and screen therapeutics for severe acute respiratory syndrome coronavirus (SARS-CoV-2) have focused on inhibiting viral cell entry by disrupting ACE2 binding with the SARS-CoV-2 spike protein. This work focuses on inhibiting SARS-CoV-2 entry through a hypothesized α5ß1 integrin-based mechanism, and indicates that inhibiting the spike protein interaction with α5ß1 integrin (+/- ACE2), and the interaction between α5ß1 integrin and ACE2 using a molecule ATN-161 represents a promising approach to treat COVID-19.

Bioorthogonal Reactions Amplify Magnetic Nanoparticles Binding and Assembly for Ultrasensitive Magnetic Resonance Sensing.

Conventional transverse relaxation time (T2)-mediated magnetic resonance sensors (MRS) that utilizing the target-induces state change of magnetic nanoparticles (MNPs) mainly suffer from low sensitivity. Recent T2-MRS that based on target-induced amount change of MNPs can achieve a higher sensitivity, but these sensors can hardly accommodate small molecules. We herein develop an ultrasensitive T2-MRS that enable the detection of small molecules based on cascade bioorthogonal reactions (BRs)-realized MNPs binding and assembly. Benefiting from rapid and highly selective cascade BRs, a single small molecule target can not only increase MNPs binding but also assembly MNPs, which greatly amplifies T2 signal for sensing based on both the state and amount change of MNPs for the first time. Our strategy is capable of sensing chlorpyrifos with a liner range of 0.1 ng/mL to 1000 ng/mL. We justify the practicability of our assay by detecting chlorpyrifos in apple and cabbage samples, whose accuracy is higher than that of enzyme linked immunosorbent assay. Our assay provides a cascade BRs-mediated MRS that can greatly broaden the use of T2-based MRS for ultrasensitive sensing trace small molecules in complex samples.

Small molecule-decorated gold nanoparticles for preparing antibiofilm fabrics.

The increase in antibiotic resistance reported worldwide poses an immediate threat to human health and highlights the need to find novel approaches to inhibit bacterial growth. In this study, we present a series of gold nanoparticles (Au NPs) capped by different N-heterocyclic molecules (N_Au NPs) which can serve as broad-spectrum antibacterial agents. Neither the Au NPs nor N-heterocyclic molecules were toxic to mammalian cells. These N_Au NPs can attach to the surface of bacteria and destroy the bacterial cell wall to induce cell death. Sonochemistry was used to coat Au NPs on the surface of fabrics, which showed superb antimicrobial activity against multi-drug resistant (MDR) bacteria as well as excellent efficacy in inhibiting bacterial biofilms produced by MDR bacteria. Our study provides a novel strategy for preventing the formation of MDR bacterial biofilms in a straightforward, low-cost, and efficient way, which holds promise for broad clinical applications.

Albumin Broadens the Antibacterial Capabilities of Nonantibiotic Small Molecule-Capped Gold Nanoparticles.

Nonantibiotic small molecule-modified gold nanoparticles (Au NPs) show great potential as an alternative for commercial antibiotics, yet their narrow antibacterial spectrum hinders the wide application in clinics. We observe that Au NPs cofunctionalized with both bovine serum albumin (BSA) and 4,6-diamino-2-pyrimidinethiol (DAPT) can generate conjugates (Au_DAPT_BSA) with progressive antimicrobial activities, including decreased minimal inhibitory concentration against Gram-negative bacteria and extended antibacterial spectrum against Gram-positive bacteria compared with DAPT-capped Au NPs (Au_DAPT). Au_DAPT_BSA induces no drug resistance and can significantly decrease the number of bacteria in the biofilms formed by Pseudomonas aeruginosa and Staphylococcus aureus. In addition, Au_DAPT_BSA exhibit in vivo healing efficiency for mice with subcutaneous abscesses caused by clinically isolated, multidrug resistant Escherichia coli or S. aureus without inducing detectable toxicity to the mammalian cells/animals. Our findings provide a new strategy for strengthening nanomaterial-based bactericides such as Au NPs, especially against drug-resistant bacterial infections.

Click Reaction-Mediated T2 Immunosensor for Ultrasensitive Detection of Pesticide Residues via Brush-like Nanostructure-Triggered Coordination Chemistry.

We develop an ultrasensitive T2-mediated immunosensor based on the coordination chemistry and Cu(I)-catalyzed 1,3-dipolar cycloaddition of azide andalkyne (CuAAC) and apply it for the detection of pesticide residues. We functionalize polyglutamic acid (PGA) on polystyrene to form a brush-like nanostructure that has a large loading capacity of Cu(II) through the coordination chemistry between PGA and Cu(II). Such a brush-like nanostructure could be used to chelate Cu(II) to modulate the CuAAC between azide-functionalized 1000 nm polystyrene (PS1000) and alkyne-functionalized 30 nm magnetic nanoparticles (MNP30), and the MNP30-PS1000 conjugate as a product of CuAAC can act as a magnetic probe in this T2-based immunosensor. This click chemistry and coordination chemistry-mediated immunosensor allows for an ultrasensitive detection for chlorpyrifos residue (0.022 ng/mL), a 58-fold enhancement compared with that of enzyme-linked immunosorbent assay (1.28 ng/mL), providing a promising platform for detection of trace small molecules.

Ag+ -Gated Surface Chemistry of Gold Nanoparticles and Colorimetric Detection of Acetylcholinesterase.

Chemical regulation of enzyme-mimic activity of nanomaterials is challenging because it requires a precise understanding of the surface chemistry and mechanism, and rationally designed applications. Herein, Ag+ -gated peroxidase activity is demonstrated by successfully modulating surface chemistry of cetyltrimethylammonium bromide-capped gold nanoparticles (CTAB-AuNPs). A surface blocking effect of long-chain molecules on surfaces of AuNPs that inhibit peroxidase activity of AuNPs is found. Ag+ ions can selectively bind on the surfaces of AuNPs and competitively destroy CTAB membrane forming Ag+ @CTAB-AuNPs complexes to result in enhanced peroxidase activity. Ag+ @CTAB-AuNPs show the highest peroxidase activity compared to similar-sized citrate-capped and ascorbic acid-capped AuNPs. Ag+ @CTAB-AuNPs can potentially develop into analyte-responsive systems and exhibit advantages in the optical sensing field. For example, the Ag+ @CTAB-AuNPs system shows an enhanced sensitivity and selectivity for acetylcholinesterase activity sensing compared to other methods.

Nanocatalyst Complex Can Dephosphorylate Key Proteins in MAPK Pathway for Cancer Therapy.

Controlling phosphorylation processes of proteins is a facile way for manipulating cell fates. Herein, a synergistic therapeutic strategy utilizing a near-infrared (NIR)-responsive nanocatalyst (NC) complex is presented, which is comprised of photoactive NC and protein phosphatase 2A (PP2A), to synergistically inhibit hyperphosphorylation of mitogen-activated protein kinase (MAPK) pathway for cancer therapy, as an example of many biological processes this approach can apply to. NIR-triggered release of PP2A specially dephosphorylates and inactivates mitogen-activated protein kinase kinase (MAP2K, also known as MEK) and extracellular regulated protein kinases (ERK) in the MAPK pathway, meanwhile, the NIR-triggered activation of NC decreases the level of intracellular adenosine triphosphate to attenuate protein phosphorylation of MEK and ERK. The synergistic therapeutics effectively suppress melanoma progression by inhibiting hyperphosphorylation of the MAPK pathway. In addition, the nanocatalyst complex reduces the risk of drug-resistance through inhibiting a rebound of RAS-GTP. The NIR-responsive nanocatalyst complex paves a novel way for cancer therapeutics.

Fe-T1 Sensor Based on Coordination Chemistry for Sensitive and Versatile Bioanalysis.

The main challenge of paramagnetic ions-mediated magnetic sensors is their relatively low sensitivity. In this study, we observe the amplification of longitudinal relaxation time (T1) signal when Fe2+ transforms into Fe3+ followed by the coordination of potassium thiocyanate (KSCN) and develop a sensitive Fe-T1 sensor based on the coordination chemistry between KSCN and Fe3+ to amplify the T1 signal for detecting a series of targets, such as hydrogen peroxide, glucose, and antigen/antibody. We justify the practicability of our assay by successfully detecting tetracycline in milk samples and hepatitis C virus in clinical samples with satisfactory accuracy. This KSCN-mediated Fe-T1 sensor not only realizes biochemical analysis and immunoassay with higher sensitivity but also retains many advantages of paramagnetic ions-mediated magnetic sensors (good stability and straightforward operation), which holds great promise for the detection of a range of targets of interest in complex samples.

Mixing-to-Answer Iodide Sensing with Commercial Chemicals.

We develop a convenient, colorimetric assay (Au/PEI) for rapid iodide (I-) determination that can be prepared facilely by mixing commercially available chemicals including tetrachloroauric acid (HAuCl4) and poly(ether imide) (PEI), and the assay can be carried out directly by adding the samples to the assay without any pretreatment and additional procedure. Au/PEI operates on the principle that I- accelerates the formation of Au NPs, which leads to a visible color change from light yellow to red for naked-eye readout with high specificity. We integrate our assay on solid devices including gel hybrids (Au/PEI/GH) and filter paper (Au/PEI paper) to satisfy the demand of point-of-care testing and justify the practicality by detecting I- in lake water that was supplemented with 10, 20, or 40 µM of I-. Au/PEI/GH with the limit of detection of 0.35 µM can satisfy the detection of drinking water based on the guidelines (1.2 µM) set by the Chinese government, and Au/PEI paper can be used even after 1 year of storage. Such assays provide a convenient and straightforward choice for routine, on-site I- tests.

Peptide-Mediated Controllable Cross-Linking of Gold Nanoparticles for Immunoassays with Tunable Detection Range.

The colorimetric immunoassay based on gold nanoparticles (AuNPs) can hardly enable simultaneous detection of multiple biomarkers in vastly different concentrations (e.g., pg/mL-µg/mL) because of its narrow dynamic range. In this work, we demonstrate an immunoassay with tunable detection range by using peptide-mediated controlled aggregation of surface modification-free AuNPs. Alkaline phosphatase (ALP) removes the phosphate group of the peptide to yield a positively charged product, which triggers the aggregation of negatively charged AuNPs and the color change of the AuNPs solution from red to blue with naked-eye readout. We design and screen 20 kinds of phosphorylated peptides to obtain a broad and controllable detection range for ALP sensing and apply them for detecting multiple inflammatory biomarkers in clinical samples. Our assay realizes straightforward, multiplexed, and simultaneous detection of multiple clinical biomarkers with tunable detection range (from pg/mL to µg/mL) in the same run and holds great potential for chemical/biochemical analysis.