RESEARCH

Screening, diagnosis, prognosis and treatment evaluation of malignant and infectious diseases often requires biomarker analyses, but restrictions imposed by current invasive or labor-intensive procedures can limit disease detection and management efforts. Noninvasive diagnostic and prognostic assays are needed to address these issues, but their development is inhibited by low biomarker concentrations, or lack of known biomarker targets, in such samples.

To address these needs, my laboratory employs advanced proteomics and engineered nanomaterial platforms to discover and validate new functionally selected disease biomarkers and to develop and validate novel assays that analyze minimally invasive samples to cover the entire spectrum of disease evaluation, including: 1) diagnosis of early disease and assessment of infectivity; 2) discrimination among specific disease stages (e.g., latent, activating, and active infections) or prognoses (e.g., treatment failure, drug resistance, or metastasis); 3) evaluation of microbial, chronic or malignant disease burden for treatment monitoring; and 4) early detection of drug resistance and toxicity, including that drug and radiation induced toxicity.

We employ biomarker function, sequence, and expression (stage-specificity and abundance) information to design assays to comprehensively profile the pathological status of an individual. We focus on diseases with unmet diagnostic/prognostic needs, and utilize nanomaterial properties to address issues that limit conventional assays. We also consider the feasibility of translating an assay into a clinical application early in the design process to improve its potential for subsequent translation.

My group utilizes nanomaterials, which offer multiple properties that can be exploited to allow disease diagnosis with biomarkers, samples, and/or methods that would not otherwise be feasible, particularly in clinical applications. We have developed several integrated procedures that utilize nanomaterials to improve the sensitivity and reliability of biomarker quantification, most of which can be readily repurposed to assay different biomarkers, including biomarkers for emerging diseases, by altering their surface properties or affinity, to reduce assay development and clinical translation effort. We have used this platform flexibility to produce TB and HIV-1 assays and to develop assays for non-tuberculous mycobacteria (NTM) and Ebolavirus species responsible for human disease.

Platform I.
Extracellular Vesicle (EV) biomarker Discovery assays

EVs are small membrane-bound vesicles secreted by all cells, circulate at high levels, and convey nucleic acids, peptide/proteins, lipids, and membrane-associated receptors and ligands to local or distant sites, where EV uptake can modify recipient cell behavior. EVs secreted by microbial pathogens or infected or malignant cells represent an excellent source of biomarkers, but technical challenges have prevented development of EV-based assays. With the advanced technologies, we have identified and validated new biomarkers for rapid pathogen differentiation (e.g. SARS-CoV-2, mycobacterial, ebolavirus, and coronavirus species), early disease diagnosis (including cancer), and/or real-time evaluation of disease response. Our unique and multidisciplinary expertise enables us to employ the characteristic properties of engineered nanodevices to improve the capture and detection of circulating and/or secreted biomarkers.

Representative Publications

  1. Ning, B., Huang, Z., Youngquist, B. M, Scott, J. W., Niu, A., Bojanowski, C., M., Zwezdaryk, K. J., Saba, N., S., Fan, J., Yin, X., Cao, J., Lyon, C. J., Li, C., Roy, C. J., Hu, T. Liposome-mediated detection of SARS-CoV-2 RNA-positive extracellular vesicles in plasma. Nature Nanotechnology. 2021. 16, 1039-1044.

  2. Sun, D., Zhao, Z., Spiegel, S., Liu, Y., Fan, J., Amrollahi, P., Lyon, C. J., Wan, M., Hu, T. Dye-free spectrophotometric measurement of nucleic acid-to-protein ratio for cell selective extracellular vesicle discrimination. Biosensors and Bioelectronics. 2021. 179: 113058.

  3. Liu, Y., Fan, J., Xu, T., Ahmadinejad, N., Hess, K., Lin, S., Zhang, J., Liu, X., Liu, X., Ning, B., Liao, Z., Hu, T. Extracellular Vesicle Tetraspanin-8 Expression Predicts Distant Metastasis in Non-Small Cell Lung Cancer after Concurrent Chemoradiation. Science Advances. 2020, 6:11, eaaz6162.

  4. Zhao, Z., Ning, B. Lyon, C., Hu, T. Extracellular vesicles as cancer liquid biopsies: from discovery, validation, to clinical application. Lab-on-a-Chip. 2019, 19(7):1114-1140.

  5. Fan, J., Wei, Q., Koay, E. J., Bernard, P.W., Kai, M., Borsoi, C., Bernard, D.W., Zhang, N., Katz, M.H., Yokoi, K., Zhao, Z., Hu, T.* Chemoresistance transmission via exosome-mediated EphA2 transfer in pancreatic cancer. Theranotics.  2018, 8(21):5986-5994.

Platform II.
Nanoplasmonic quantification of pathogen-derived extracellular vesicles

To address the need in rapid and isolation-free quantification of circulating EV for clinical appliations, we have developed a novel, streamlined procedure in which EVs are captured directly from serum/plasma with an antibody recognizing a ubiquitous EV membrane protein and then hybridized with antibody-conjugated gold nanorods (AuR) and nanospheres (AuS) that respectively bind a common and a disease-specific EV membrane protein. Only EVs secreted by diseased cells bind both probes to form AuR-EV-AuS complexes and plasmons that markedly shift the spectrum and intensity of scattered light to distinguish target EVs from AuR-EV complexes that emit weak signal at a different wavelength.

 

The above assays are designed to rapidly read biomarkers on the surface of EVs, since analysis of biomarkers carried as EV cargoes normally requires the isolation of total or target EV lysates, which can reduce assay sensitivity and specificity due to the variable purity and background of EV fractions generated for these assays. To address this issue, my group is developing a purification-free assay for such analyses in which customized liposomes containing molecular beacons are directed to bind target EV populations through specific recognition of an EV surface marker, allowing EV cargo biomarkers to be analyzed directly from serum or plasma samples

Representative Publications

1. Liang K., Liu, F., Fan, J., Sun, D., Bernard, D. W., Li, Y., Katz, M. H., Koay, E. J., Zhao, Z., Hu, T. Nanoplasmonic Quantification of Tumor-derived Extracellular Vesicles in Plasma Microsamples for Diagnosis and Treatment Monitoring. Nature Biomedical Engineering. 2017, 1:0021.

2. Rodrigues, M., Richards, N., Ning, B., Lyon, C., Hu, T. Rapid lipid-based normalization of membrane protein expression on extracellular vesicles in complex biological samples. Nano Letters. 2019, 19, 11, 7623-7631

3. Amrollahi P., Rodrigues M., Lyon, C. J., Goel, A., Han, H., Hu, T. Ultra-sensitive automated profiling of EpCAM expression on tumor-derived extracellular vesicles. Frontiers in Genetics. 2019, 10:1273.

4. Sun, D., Lyon, C., Hu, T. Simulation-directed amplifiable nanoparticle enhanced quantitative scattering assay under low magnification darkfield microscope. Journal of Material Chemistry B 2020, 8: 5416-5419.

Platform III. 
Peptidomic quantification of pathogen antigens for a personalized diagnosis

Circulating levels of pathogen-derived proteins can serve as useful biomarkers for infections that occur at unknown or inaccessible anatomical sites, but may be detected with poor sensitivity and specificity by standard immunoassays due to masking effects and cross-reactivity with proteins of related pathogens. Mass spectrometry (MS)-read immunoassays that detect peptides from such biomarkers can resolve these issues, but there are no workflows to identify such candidate peptide biomarkers or to distinguish weak biomarker signal from MS background noise. To address these issues, we employed nanomaterials and proteomics to develope workflows to select the high-performance peptide biomarkers and to reject false-positive signals using MS peak criteria, leading to an accurate detection of the active tuberculosis, EBLOA and HIV diseases. Mass spectrometry represents an attractive means of identifying serum biomarkers derived from bacterial species, since it can discriminate peptides derived from homologous proteins, such as virulence factors, which are expressed by multiple pathogen species. This workflow can be applied to other pathogens to facilitate sensitive and specific diagnosis of infectious diseases.

Representative Publications

  1. Liu, C., Zhao, Z., Fan, J., Lyon, C. J., Wu, H., Nedelokv, D., Zelazny, A.M., Olivier, K.N., Cazares, L.H., Holland, S.H., Graviss, E. A., Hu, T. Quantification of Circulating M. tuberculosis Antigens for Rapid Diagnosis and Real-time Treatment Monitoring. Proc. Natl. Acad. Sci. U S A.  2017, 114(15):3969-3974.

  2. Liu, C., Lyon, C. J., Deng, Z., Walters, E., Li, Y., Zhang, L., Hesseling, A., Graviss, E. A., Hu, T. Clinical evaluation of a blood assay to diagnose paucibacillary tuberculosis via bacterial antigens. Clinical Chemistry 2018, 64(7): 1

  3. Mao, L., Lacourse, S., Kim, S., Ning, B., Bao, D., Fan, J., Sun, Z., Nackman, S., Mitchelle, C., Hu, T. Evaluation of a blood-based antigen test for tuberculosis in HIV-exposed children younger than 5 years. BMC Medicine. 2021, 19:113.

  4. Shu, Q., Kenny, T., Fan, J., Lyon, C., Cazares, L., Hu. T. Species-specific Quantification of Circulating Ebolavirus Burden using VP40-derived Peptide Variants. PLOS Pathogens. 2021. Accepted.

Platform IV.
Ultra-sensitive CRISPR-based Detection of Pathogen Species 

Our research on COVID-19 has been devoted to developing more sensitive assays that to improve diagnosis, screening, and treatment evaluation efforts. Improved assay methods are urgently needed since current assays are known to miss significant numbers of cases and appear to falsely identify viral clearance in a subset of patients that subsequently develop disease recurrence. My research team has developed a rapid, ultrasensitive COVID-19 diagnostic assay that is more sensitive, and requires less specialized equipment, than the current gold standard assay. We have applied to the FDA requesting that Tulane-affiliated hospitals be allowed to use this assay for general COVID-19 diagnosis under Emergency Use Authorization regulations. My group is now validating the ability of ability of this assay to diagnose COVID-19 using other diagnostic samples that can be more readily collected than nasopharyngeal swabs used in most current assays or which can provide different diagnostic or prognostic information. We are also refining this assay approach to be read on inexpensive hand-held devices that are suitable for large scale deployment in community-based screening efforts.

Representative Publications

  1. Huang, Z., Ning, B., Yang, H., Youngquist, B., Niu, A., Lyon, C., Beddingfield, B., Fears, A., Monk, C., Murrell, A., Bilton, S., Linhuber, J., Norton, E., Dietrich, M., Lai, W., Scott, J., Yin, X., Rappaport, J., Robinson, J., Saba, N., Roy, C., Zwezdaryk, K., Zhao, Z., Hu, T.* Sensitive tracking of viral RNA in plasma through all stages of SARS-CoV-2 infection. J. Clinical Investigation. 2021, 131(7):e146031.

  2. Huang, Z., Tian, D., Liu, Y., Lin, Z., Lyon, C., J., Lai, W., Fusco, D., Drouin, A., Yin, X., Hu, T.*, Ning, B.* Ultra-sensitive and high-throughput CRISPR-powered COVID-19 diagnosis. Biosensor and Bioelectronics 2020. 164: 112316.

  3. Niu, A., Ning, B., Socola, F., Safah, H., Reynolds, T., Ibrahim, M., Safa, F., Alfonso, T., Luk, A., Mushatt, D., Hu, T., Saba, N. S. COVID-19 in Patients with Hematological Malignancies: High False Negative Rate with High Mortality. Blood. 2020, 136 (1): 6-7.

Platform V.
Development of Portable Diagnostic Devices for Resource-limited Areas

These assays utilize streamlined workflows suitable for clinical applications and employ FDA-approved MS systems, bench-top dark-field microscope or plate reader to facilitate clinical translation. Our group is also dedicated to translating our findings into applications suitable for resource-limited areas, and is currently developing multiple portable and inexpensive systems to quantify our peptide, nucleic acid and extracellular vesicle targets.

Representative Publications

  1. Ning, B., Yu, T., Zhang, S., Huang, Z., Tian, D., Lin, Z., Niu, A., Golden, N., Hensley, K., Threeton, B., Lyon, C. J., Yin, X., Saba, N., Rappaport, J., Wei, Q., Hu, T.* A smartphone-read ultrasensitive and quantitative saliva test for COVID-19. Science Advances. 2021. 7(2): eabe3703.

  2. Sun, D., Hu, T.* A low cost mobile phone-based dark-field microscope for nanoparticle-based quantitative studies. Biosensors and Bioelectronics 2017, 15;99:513-518.

  3. Zou, R., Cao, W., Chong, L., Hua, W., Xu, H., Mao, Y., Page, J., Shi, R., Xia Y., Hu, T., Zhang, W., Ouyang, Z. Point-of-Care Tissue Analysis Using Miniature Mass Spectrometer. Analytical Chemistry, 2018, 91, 1157-1163.

Platform VI.
Nanotechnology-based sensing platforms

To manufacture sensing devices with the precision and consistency required for clinical use, we applied micro-/nano-fabrication technology developed and established over the past 50 years in the semiconductor industry. Applying nanotechnology to the biosciences offers an enormous advantage due to immediate access to an array of sophisticated tools. Scalability, precision, and reproducibility are valuable characteristics of the micro-machining processes that can be translated into clinical applications. My research team has been developing integrated nanomaterial-based microsystems, semiconductor chips and nanotechnologies for imaging, sensing, and regulating cellular processes critical to healthcare, environmental, and defense applications. Nano-Micro scale science, information, and biomedicine are integrative components of my research, and are used in combination with advanced engineering tools to facilitate biomedical studies and develop robust diagnostics for global health initiatives.

Representative Publications

  1. Sun, D., Lyon, C., Hu, T.* Simulation-directed amplifiable nanoparticle enhanced quantitative scattering assay under low magnification darkfield microscope. Journal of Material Chemistry B 2020, 8: 5416-5419.

  2. Deng, Z., Zhao, Z., Basilio, J., Ye, Y., Mann, K., Fu, J., Gu, Y., Wu, X., Chiao, P., Hu, T.* Nanotrap-enabled quantification of KRAS-induced peptide hydroxylation in blood for cancer early detection. Nano Research. 2019, 12(6): 1445-1452.

  3. Wu, H. J., Li, Y., Fan, J., Deng, Z., Hu, Z., Liu, X., Graviss, E. A., Ma, X., Hu, T.* Antibody-Free Detection of M. tuberculosis Antigen Using Customized Nanotraps. Anal. Chem. 2014; 86 (4), pp 1988–1996. DOI: 10.1021/ac4027669.

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Platform VII.
Nanopore-Enabled Peptidomic Analysis and Biomarker Discovery

Low molecular weight (LMW) peptides, rather than bulky and abundant proteins, are more viable sources of biomarkers; disease-associated peptides (secreted by cells, shed from their surface or otherwise released) are more likely to enter the bloodstream where we can quickly and easily survey them as part of an early detection strategy for health care. However, despite great investment of resources, efforts to identify serological protein/genetic biomarkers for most diseases have met with limited success. Most current techniques can only capture high molecular weight (HMW) proteins, whereas the real information encoded within low-abundance, small peptides remains elusive, hindering our true understanding of the immunologic process of disease malignance and our ability to detect its early stage with any real accuracy. Our research design addresses current conceptual and technical gaps. We have developed a tunable, nanopore-based platform to effectively fractionate peptides from blood samples with little to no sample processing. With the superior capillary adsorption of nanopore films to peptides via engineering of the nanopore’s physico-chemical properties (e.g., pore size, pore structure, surface affinity), we have been able to enrich these LMW peptides from serum and preserve their integrity. By coupling this platform to advanced mass spectrometry technology and customized biostatistics methods, our discoveries have enormous promise for rapid translation to the clinic, particularly by offering an inexpensive and precise platform for disease diagnosis in vulnerable populations. The examples below highlight our contribution to this field.

Representative Publications

  1. Liu, Y., Gu, Y., Lyon, C. J., Fan, J., Koay, E., Katz, M., Han, H., Von Hoff, D., Hu, T.  Circulating levels of hydroxylated bradykinin function as an indicator of tissue HIF-1α expression. Science Bulletin. 2020,65(18): 1570-1579.

  2. Deng, Z., Zhao, Z., Basilio, J., Ye, Y., Mann, K., Fu, J., Gu, Y., Wu, X., Chiao, P., Hu, T. Nanotrap-enabled quantification of KRAS-induced peptide hydroxylation in blood for cancer early detection. Nano Research. 2019, 12(6): 1445-1452.

  3. Wu, B., Ouyang, Z., Lyon, C. J., Zhang, W., Clift, T., Bone, C., Zhao, Z., Kimata, J., Hu, T. Plasma C4b peptide levels and HLA-B*57 genotype are associated with spontaneous HIV suppression in HIV-1-infected patients. ACS Infect. Dis. 2017, 3 (12): 880–885.

  4. Book: Peptidomics of Cancer-Derived Enzyme Products, The Enzymes: Volume 42 edited by Dr. Tony Hu & Dr. Fuyu Tamanoi, 2017 Academic Press/Elsevier.

  5. Li, Y., Li, Y., Chen, T., Kuklina, A. S., Bernard, P., Esteva, F. J., Shen, H., Hu, T. Circulating Proteolytic Products of Carboxypeptidase N for Early Detection of Breast Cancer. Clin. Chem. 2014, 60(1): 233-242. PMID: 24146311.

Funding