Publications

Despite major advances in HIV testing, ultrasensitive detection of early infection remains challenging, especially for the viral capsid protein p24, which is an early virological biomarker of HIV-1 infection. Here, To improve p24 detection in patients missed by immunological tests that dominate the diagnostics market, we show a click chemistry amplified nanopore (CAN) assay for ultrasensitive quantitative detection. This strategy achieves a 20.8 fM (0.5 pg/ml) limit of detection for HIV-1 p24 antigen in human serum, demonstrating 20~100-fold higher analytical sensitivity than nanocluster-based immunoassays and clinically used enzyme-linked immunosorbent assay, respectively. Clinical validation of the CAN assay in a pilot cohort shows p24 quantification at ultra-low concentration range and correlation with CD4 count and viral load. We believe that this strategy can improve the utility of p24 antigen in detecting early infection and monitoring HIV progression and treatment efficacy, and also can be readily modified to detect other infectious diseases.

Abstract Perfluorooctanoic acid (PFOA) has been one of the most common perfluorochemicals, which are globally pervasive contaminants that are persistent, bioaccumulative, toxic, and have adverse impacts on human health. The highest concentration of PFOA occurs in the blood, where it strongly binds to human serum albumins (HSA). Thus, a method to reverse the HSA-PFOA binding is critical to help facilitate the faster elimination of PFOA from the body to minimize its toxicological effects. Inspired by the remediation effect of cyclodextrin (CD) to PFOA through host-guest interactions, herein, by elucidating inter-molecular interactions using a nanopore sensor, we demonstrated in vitro reversal of the binding of PFOA to HSA using γ-cyclodextrin (γ-CD). The competition behavior for the complexation of PFOA between HSA and γ-CD was discussed in combination with in situ nanopore current recording and nuclear magnetic resonance (NMR) characterization. The present work not only demonstrates the potential therapeutic application of γ-CD for PFOA removal from human blood, but also provides an emerging method for investigating interactions between organic compounds and proteins.

DNAs have been used as probes for nanopore sensing of noncharged biomacromolecules due to its negative phosphate backbone. Inspired by this, we explored the potential of diblock synthetic polyelectrolytes as more flexible and inexpensive nanopore sensing probes by investigating translocation behaviors of PEO-b-PSS and PEO-b-PVBTMA through commonly used alpha-hemolysin (α-HL) and Mycobacterium smegmatis porin A (MspA) nanopores. Translocation recordings in different configurations of pore orientation and testing voltage indicated efficient PEO-b-PSS translocations through α-HL and PEO-b-PVBTMA translocations through MspA. This work provides insight into synthetic polyelectrolyte-based probes to expand probe selection and flexibility for nanopore sensing.

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.

Diagnosis of pediatric tuberculosis (TB) is often complicated by its nonspecific symptoms, paucibacillary nature, and the need for invasive specimen collection techniques. However, a recently reported assay that detects Mycobacterium tuberculosis virulence factors in serum can diagnose various TB manifestations, including paucibacillary TB cases, in adults with good sensitivity and specificity. Diagnosis of pediatric tuberculosis (TB) is often complicated by its nonspecific symptoms, paucibacillary nature, and the need for invasive specimen collection techniques. However, a recently reported assay that detects Mycobacterium tuberculosis virulence factors in serum can diagnose various TB manifestations, including paucibacillary TB cases, in adults with good sensitivity and specificity. The current study examined the ability of this M. tuberculosis biomarker assay to diagnose pediatric TB using archived cryopreserved serum samples drawn from children ≤18 years of age who were screened for suspected TB as part of a prospective population-based active surveillance study. In this analysis, any detectable level of either of the M. tuberculosis virulence factors CFP-10 and ESAT-6 was considered direct evidence of TB. Serum samples from 105 children evaluated for TB (55 TB cases and 50 close contacts without TB) were analyzed. The results of this analysis yielded sensitivity of 85.5% (95% confidence interval [CI], 73.3 to 93.5). Similar diagnostic sensitivities were observed for culture-positive (87.5%; 95% CI, 67.6 to 97.3) and culture-negative (83.9%; 95% CI, 66.3 to 94.5) TB cases and for culture negative pulmonary (77.8%; 95% CI, 40.0 to 97.2) and extrapulmonary (86.4%; 95% CI, 65.1 to 97.1) TB cases. These results suggest that serum biomarker analysis holds significant promise for rapid and sensitive diagnosis of pediatric TB cases, including extrapulmonary or paucibacillary TB cases. The ability to use frozen samples for this analysis should also permit assays to be performed at central sites, without a requirement for strict timelines for sample analysis.

The coronavirus disease (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread into a global pandemic. Early and accurate diagnosis and quarantine remain the most effective mitigation strategy. Although reverse transcriptase polymerase chain reaction (RT-qPCR) is the gold standard for COVID-19 diagnosis, recent studies suggest that nucleic acids were undetectable in a significant number of cases with clinical features of COVID-19. Serologic assays that detect human antibodies to SARS-CoV-2 serve as a complementary method to diagnose these cases, as well as to identify asymptomatic cases and qualified convalescent serum donors. However, commercially available enzyme-linked immunosorbent assays (ELISA) are laborious and non-quantitative, while point-of-care assays suffer from low detection accuracy. To provide a serologic assay with high performance and portability for potential point-of-care applications, we developed DNA-assisted nanopore sensing for quantification of SARS-CoV-2 related antibodies in human serum. Different DNA structures were used as detection reporters for multiplex quantification of immunoglobulin M (IgM) and immunoglobulin G (IgG) antibodies against the nucleocapsid protein of SARS-CoV-2 in serum specimens from patients with conformed or suspected infection. Comparing to a clinically used point-of-care assay and an ELISA assay, our technology can reliably quantify SARS-CoV-2 antibodies with higher accuracy, large dynamic range, and potential for assay automation.

Alzheimer’s disease and other neurodegenerative disorders are becoming more prevalent as advances in technology and medicine increase living standards and life expectancy. Alzheimer’s disease is thought to initiate development early in the patient’s life and progresses continuously into old age. This process is characterized molecularly by the amyloid hypothesis, which asserts that self-aggregating amyloid peptides are core to the pathophysiology in Alzheimer’s progression. Precise quantification of amyloid peptides in human bodily fluid samples (i.e. cerebrospinal fluid, blood) may inform diagnosis and prognosis, and has been studied using established biosensing technologies like liquid chromatography, mass spectrometry, and immunoassays. However, existing methods are challenged to provide single molecule, quantitative analysis of the disease-causing aggregation process. Ultra-sensitive nanopore biosensors can step in to fill this role as a dynamic mapping tool. The work in this paper establishes characteristic signals of β-amyloid 40 monomers, oligomers, and soluble aggregates, as well as a proof-of-concept foundation where a biological nanopore biosensor is used to monitor the extent of in vitro β-amyloid 40 peptide aggregation at the single molecule level. This foundation allows for future work to expand in drug screening, diagnostics, and aggregation dynamic experiments.

Non-sputum methods are urgently needed to improve tuberculosis diagnosis and treatment monitoring in children. This study evaluated the ability of a serum assay quantifying a species-specific peptide of the Mycobacterium tuberculosis CFP-10 virulence factor via nanotechnology and matrix-assisted laser desorption ionization time-of-flight mass spectrometry to diagnose tuberculosis in HIV-infected and HIV-uninfected infants.

Nanopore technology holds remarkable promise for sequencing proteins and peptides. To achieve this, it is necessary to establish a characteristic profile for each individual amino acid through the statistical description of its translocation process. However, the subtle molecular differences among all twenty amino acids along with their unpredictable conformational changes at the nanopore sensing region result in very low distinguishability. Here we report the electrical sensing of individual amino acids using an α-hemolysin nanopore based on a derivatization strategy. Using derivatized amino acids as detection surrogates not only prolongs their interactions with the sensing region, but also improves their conformational variation. Furthermore, we show that distinct characteristics including current blockades and dwell times can be observed among all three classes of amino acids after 2,3-naphthalenedicarboxaldehyde (NDA)- and 2-naphthylisothiocyanate (NITC)-derivatization, respectively. These observable characteristics were applied towards the identification and differentiation of 9 of the 20 natural amino acids using their NITC derivatives. The method demonstrated herein will pave the way for the identification of all amino acids and further protein and peptide sequencing.