Nanotechnology is a promising tool for improving diagnosis, assessing treatment efficacy and designing personalized therapies with minimal invasiveness. Our research team is developing integrated mesoporous material-based microsystems, semiconductor chips and nanotechnologies for imaging, sensing and regulating cellular processes critical to health care, and environmental and defense applications.

Our laboratory’s current research focuses on developing tools and methods to understand and regulate complex biological networks that are critical to the development of disease, and developing nano-micro-fabricated sensors for biomaterial characterization that can be used to improve precancerous lesion detection. Nano-micro-scale science, information and biomedicine are integrative components of our research, and are used in combination with advanced engineering tools to facilitate biomedical studies and develop robust diagnostics for global health initiatives.

I. Nanopore-Enabled Peptidomic Analysis and Biomarker Discovery

Disease development may be characterized by molecular changes (e.g., enzymatic activities, protein shedding, extracellular matrix remodeling, etc.) in diseased cells or tissues prior to noticeable physiological symptoms. If particular biomolecules associated with disease progression in microenvironment can be detected in a patient’s blood, they may be used as potential biomarkers to inform clinical decisions throughout the continuum of health care. 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.

Blood-based biomarkers are amenable to the development of user-friendly diagnostics, partly due to their accessibility. They could also have an important place in early cancer detection strategies, as the immune system plays an active role in the tumor microenvironment by regulating tumor progression. Throughout the processes of cell seeding, quiescence, replication and eventual progression of disease, host factors are often deployed to survey for the presence of tumors. Peptides released into the blood by the immune system have been recognized as very good indicators of impending or occurring malignancy. However, despite great investment of resources, efforts to identify serological protein/genetic biomarkers for most diseases (e.g., Cancer diseases) have met with limited success. Current techniques can largely 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, by offering an inexpensive and precise platform for disease diagnosis in the more vulnerable population.

II. Circulating Proteolytic Products of Tumor-Resident Proteases for Early Detection of Cancer

Circulating peptides have been recognized as useful signatures that can be traced to cancer-specific metabolic or post-translational modification events at early-stage tumor progression. However, efforts to identify serological peptide biomarkers have met with limited success, hindering understanding of the biological process of cancer development and our ability to detect it early with any real accuracy. It is known that serum peptides can provide accurate class discrimination between patients and healthy controls. Yet, it remains unclear whether this complex peptidome may provide a robust correlate of certain biological events occurring in the entire organism. To address these technical and conceptual challenges, we have established “Nanotrap” to effectively fractionate blood peptides with little to no sample processing. By coupling this technique to advanced mass spectrometry, we can bypass the limitation of current proteomic technologies, by “amplifying” the amount of small peptides extracted from blood samples, without using immunoaffinity agents. The spectra for these species would otherwise be clouded by larger and more abundant serum proteins. Such an enrichment, performed on a high-throughput platform, enables the elimination of confounding factors to signal-to-noise optimization, and instead allows us to focus on analyzing true disease signatures. More importantly, our studies do not solely focus on the discovery and validation of novel biomarkers.

We are the first to demonstrate a link between the activity of Carboxypeptidase N (CPN) for breast cancer and prolyl-4-hydroxylase (P4H) for pancreatic cancer within tumor sites and the cleavage patterns of their catalytic substrates or their post-translational modification product in blood. Our cutting-edge nanotechnologies coupled with advanced mass spectrometry and customized biostatistical analysis facilitated the functional mechanism-driven peptide biomarker studies for revealing the early events associated with the signature mutations or pathways in tumor progression, leading to abnormal biological responses in tumor cells or its microenvironment prior to noticeable physiological symptoms.

III. Exosome Isolation and Analysis System

As extracellular vesicles, the exosomes secreted by cells are circulating in body fluids. Some of those exosomes have a size range from 30 to 100 nm; are recognized as intercellular communication vehicles, which are for supporting or interfering with physiological process and transferring biomolecules, including proteins and nucleic acids (NAs), such as messenger RNAs (mRNAs), microRNAs (miRNAs) and non-coding RNA (ncRNA).

Recent research studies show that exosomes play an important role in biological functions; and this is potentially a new disease biomarker in clinical applications, such as point-of-care (POC) diagnostics and personalized therapeutics. The proteins and nucleic acids in exosomes are much richer than those circulating in body fluids. However, the concentration of exosomes is very low in body fluids. We developed an ultrasensitive plasmonic platform for fast and quantitative detection of in vitro and in vivo derived exosomes. The dark field plasmonic counts based on  specific antibodies conjugated gold nanoparticles (GNPs) are used to quantify exosome amounts for pancreatic cancer in ten microliters of cell culture media, murine model serums, and clinical human serums, respectively. Implementing this technique can provide a fast and useful tool as quantitative analysis technique in the clinic whether it is for diagnosis, monitoring or therapeutic purposes as well as for the understanding of the role of exosomes in cancer progression. 

IV. Silicon Nanodisk for Ultrasensitive Peptide Detection

Tuberculosis (TB) infection caused by Mycobacterium tuberculosis (MTB) infection poses a significant global health challenge, not only to the patients severely affected, but also to the wider international community. Diagnostic criteria for TB are based on a host of scoring algorithms and tests, e.g., symptom presentation, chest radiography, tuberculin skin test, etc. While these have provided some relief, they are not without significant limitations, thus, driving the need for more sensitive, rapid and effective diagnostic means.

In order to address the current limitations in clinical management of pediatric TB, we have developed a rapid and quantitative diagnostic and monitoring platform for active TB wherein porous silicon nanodisks (referred to as “pSiND”), loaded with target biomarkers, are used for identification of disease signatures by mass spectrometry (MS). Using this new biomarkers detection modality, pSiND-MS, we could detect and quantify two TB-specific blood-born biomarkers (CFP-10 and ESAT-6) at extremely low concentrations (1.0 fmol), which serve as signs of bacterial infection in advance of physiological manifestations observable by conventional protocols. Characteristics of ESAT-6 and CFP-10, i.e., antigens secreted by actively proliferating MTB, make them ideal biomarkers for active TB diagnosis and candidates for TB vaccine development. We used this approach to distinguish adult patients with active TB from those with latent tuberculosis infection, or healthy volunteers. Also, based on multiplex detection and quantification by pSiND-MS, we differentiated pediatric TB-infected patients (n=36) from non-TB children (n=35) according to the peptidic patterns of CFP-10 and ESAT-6. Our categorization of patient samples matched clinical designations at 100% specificity and 94.4% sensitivity. Just as important, we could render accurate diagnoses within one hour of sample-to-answer processing rather than wait the typical 4-6 weeks. The pSiND-MS technology platform has an added advantage in that high-throughput and accurate mass spectrometry has become a virtually essential technology for clinical diagnosis in many parts of the world.