High Sensitivity Measurements

Microbial Detection

Disease diagnostics, medical therapeutics, environmental analysis, and biowarfare defense depend heavily on the quantitative and qualitative information gained from DNA and RNA analysis. Our lab employs DNA and RNA hybridization assays to detect and aid in the diagnosis of infectious diseases in environmental and clinical samples. These tasks are achieved by chemically functionalizing the surface of polystyrene beads with nucleic acid probes that serve as individual sensors for the detection of ribosomal RNA using an optical microarray platform, allowing for high density arrays.

Cyanobacteria

Freshwater cyanobacteria, also known as blue-green algae, have the potential to rival the serious effects seen with marine harmful algal blooms (HABs), also known as red tides. The ability of certain species of cyanobacteria to cause water blooms, in which toxins are released in freshwater eutrophic environments, highlights the need to effectively monitor the proliferation of cyanobacteria for public health measures and environmental integrity. Encoded polystyrene microspheres functionalized with specific nucleic acid sequences for Anabaena, Cylindrospermopsis and Microcystis were used to develop an optical microarray platform for the multiplexed detection of these three freshwater cyanobacteria.

cyano

Figure 1 illustrates the multiplexed optical array. Beads functionalized with species-specific capture probes are randomly distributed onto the array. An encoding image (a) is used to determine the location of each bead type, while a background image (b) and a hybridization image (c) are used to determine positive signal in the presence of target analyte(s).

Single Molecule Assays in Early Detection of Infectious Disease

The potential for early diagnosis of infectious diseases relies on the ability to detect ultra low concentrations of circulating pathogens and related biomarkers such as cytokines, host immunoglobulins, and DNA/RNA. Infectious diseases are a leading cause of morbidity and mortality world-wide. Thus, it is important to detect infection at its earliest stage in order to provide effective treatment and to control disease spread.

Dengue virus, a 50 nm arthropod-borne positive-sense RNA virus, is proving to be one of the most important emerging infectious diseases currently affecting more than 100 countries worldwide,1 with an estimated 3 billion people at risk of infection.2 Recent studies have shown increased diagnostic efficacy when combining confirmatory assays that detect RNA, virus, or non structural protein 1 (NS1) with serological assays, which detect anti-Dengue IgM/IgG.3 While there are several detection methods for Dengue Virus, such as virus isolation and nucleic acid detection, methods that provide more accurate detection are more costly, labor intensive and require either 1-2 weeks or 1-2 days for confirmatory results. Our lab is developing methods for the detection of circulating intact viral particles and host immunoglobulins that correlate with Dengue infection. Immunoglobulins are circulating antibodies that appear in response to foreign antigens and are part of the humoral immune response. Upon infection by bacteria or virus, immunoglobulins are secreted by B cells to recognize specific target epitopes. We aim to provide an alternative to direct viral detection, in which we detect anti-Dengue immunoglobulins (IgG and IgM) in the febrile stage, and provide insight into primary vs. secondary infection.

When pathogens enter the body they induce a variety of host-defenses such as cytokines. Cytokines, signaling proteins secreted by numerous cells, play an important role in promoting and regulating the immune response. The presence of different cytokines, and the pattern of their secretion, may indicate an infection by a particular pathogen. It has been reported that serum concentrations of different cytokines are indicative of certain infections such as sepsis, HIV-infection, tuberculosis, malaria etc.4-7 when compared to healthy controls. Thus, the determination of a broad set of cytokines can serve as a fingerprint for a particular disease.

Secreted cytokines can be quantified with bioassays, enzyme-linked immunosorbent assays (ELISA), radioactive immunosorbent assays, and microarrays. These methods typically measure proteins at concentrations above 1 pM.8 Some cytokines can reach this level only when disease is at an advanced stage. In our laboratory, we are developing single molecule, ultra-sensitive (fM to aM), assays capable of detecting and quantifying cytokines at the earliest possible level of infection. Detection of cytokines at significantly lower levels than is currently possible will allow for earlier detection of infectious diseases and may lead to treatments that avoid disease progression.

Breast Cancer Diagnostics

It has been estimated that in 2012 approximately 200,000 new cases of breast cancer will be diagnosed in the United States, resulting in approximately 40,000 deaths.9 The diagnosis of breast cancer (and virtually every cancer) at earlier stages is correlated with an increase in survival rates.10 Mammography is a powerful imaging technique for tumor detection; however, it lacks the ability to decipher benign from cancerous tumors, is unable to detect tumors smaller than 1mm,11 misses approximately 20% of breast cancers potentially present at the time of screening, and has an 8-10% false positive rate.12 These drawbacks lead to inaccurate patient diagnosis, which can allow potentially fatal disease progression, or in the cases of over-treatment, unnecessary physical and emotional trauma.13 ELISA, the most common immunoassay for measuring proteins from breast tumors, excised samples, and serum, have a lower detection limit of ~1-10 pM,14 which is not sensitive enough to measure low abundance proteins that could aid in the early and reliable diagnosis of cancer. There is a clear need to develop techniques capable of detecting biomarkers specific for breast cancer that will enable earlier diagnosis of disease, prediction of patient outcome, and improve therapeutic efficacy in a non-invasive manner. Our goals are to utilize the ultrasensitive single molecule techniques developed in our laboratory to discover new biomarkers, both proteins and miRNAs, that meet these requirements so that a simple blood test can be implemented.

Proteins

Two of the most important features of a sensitive biomarker are that it must be both specific and selective for cancer and, ideally, for a specific type of tumor. Several proteins, including cancer antigen 15-3 (CA 15-3), cancer antigen 27-29 (CA 27-29), and carcinoembryonic antigen (CEA), are currently screened in the serum of breast cancer patients for clinical use. Unfortunately, due to an inherent lack of specificity and sensitivity associated with these biomarkers, they are useful primarily for monitoring only the latter stages of disease and not for detection of primary disease or relapse.13-16 Digital ELISA has proven to be effective for the detection of several different types of proteins in serum, including prostate specific antigen (PSA) for monitoring the recurrence of prostate cancer after radical prostatectomy,17 as well as tumor necrosis factor-alpha (TNF-α) and interleukin 6 (IL-6) for monitoring therapeutic efficacy in Crohn's disease.14,18 The ability to identify and measure proteins specific to breast cancer but occur at levels that are not measurable in blood using standard methods could enable earlier detection of disease and improve therapeutic efficacy. The goals for this portion of the project are to identify proteins that possess these characteristics and to utilize the power of digital ELISA to produce protein fingerprints to create a more sensitive and specific blood test for breast cancer detection and monitoring.

MicroRNA

MicroRNAs (miRNAs) are short (18-24 nucleotide), non-coding RNAs that regulate gene expression. Once incorporated into the RNA Induced Silencing Complex (RISC), miRNAs hybridize with the 3’UTR of messenger RNAs (mRNA) to either cleave or translationally repress the expression of genes. In many diseases, miRNAs are differentially expressed in tissues, blood, and other bodily fluids. Aberrant miRNA expression in cancer leads to the suppression of tumor suppressor genes or over-expression of oncogenes.19 Previous research has shown that miRNA expression can be used to classify the stages and different molecular subtypes of breast cancer.20,21 Both the accessibility and stability of miRNAs in the bloodstream make them potential candidates for minimally invasive breast cancer biomarkers. The goal of this project is to develop single molecule miRNA assays using the optical microarray platform established in our laboratory to capture and quantify miRNAs in serum at ultra-low concentrations for early breast cancer detection, monitoring of disease progression, detection of recurrence, and improvement of therapeutic efficacy. 22,23

References

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2.         Malavige, G. N.; Fernando, S.; Fernando, D. J.; Seneviratne, S. L., Dengue viral infections. Postgraduate Medical Journal 2004, 80 (948), 588-601.
3.         Fry, S. R.; Meyer, M.; Semple, M. G.; Simmons, C. P.; Sekaran, S. D.; Huang, J. X.; McElnea, C.; Huang, C.-Y.; Valks, A.; Young, P. R.; Cooper, M. A., The Diagnostic Sensitivity of Dengue Rapid Test Assays Is Significantly Enhanced by Using a Combined Antigen and Antibody Testing Approach. PLoS Negl Trop Dis 2011, 5 (6), e1199.
4.         Laborada, G.; Rego, M.; Jain, A.; Guliano, M.; Stavola, J.; Ballabh, P.; Krauss, A. N.; Auld, P. A. M.; Nesin, M., Diagnostic value of cytokines and C-reactive protein in the first 24 hours of neonatal sepsis. Am J Perinatol 2003, 20 (8), 491-501.
5.         Haissman, J. M.; Vestergaard, L. S.; Sembuche, S.; Erikstrup, C.; Mmbando, B.; Mtullu, S.; Lemnge, M. M.; Gerstoft, J.; Ullum, H., Plasma Cytokine Levels in Tanzanian HIV-1-Infected Adults and the Effect of Antiretroviral Treatment. JAIDS Journal of Acquired Immune Deficiency Syndromes 2009, 52 (4), 493-497 10.1097/QAI.0b013e3181b627dc.
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7.         Lyke, K. E.; Burges, R.; Cissoko, Y.; Sangare, L.; Dao, M.; Diarra, I.; Kone, A.; Harley, R.; Plowe, C. V.; Doumbo, O. K.; Sztein, M. B., Serum Levels of the Proinflammatory Cytokines Interleukin-1 Beta (IL-1β), IL-6, IL-8, IL-10, Tumor Necrosis Factor Alpha, and IL-12(p70) in Malian Children with Severe Plasmodium falciparum Malaria and Matched Uncomplicated Malaria or Healthy Controls. Infection and Immunity 2004, 72 (10), 5630-5637.
8.         Giljohann, D. A.; Mirkin, C. A., Drivers of biodiagnostic development. Nature 2009, 462 (7272), 461-4.
9.         Siegel, R.; Naishadham, D.; Jemal, A., Cancer statistics, 2012. CA: A Cancer Journal for Clinicians 2012, 62 (1), 10-29.
10.       Effects of chemotherapy and hormonal therapy for early breast cancer on recurrence and 15-year survival: an overview of the randomised trials. The Lancet 2005, 365 (9472), 1687-1717.
11.       Rice, J., Metastasis: The rude awakening. Nature 2012, 485 (7400), S55-7.
12.       Taplin, S.; Abraham, L.; Barlow, W. E.; Fenton, J. J.; Berns, E. A.; Carney, P. A.; Cutter, G. R.; Sickles, E. A.; Carl, D.; Elmore, J. G., Mammography facility characteristics associated with interpretive accuracy of screening mammography. J Natl Cancer Inst 2008, 100 (12), 876-87.
13.       Duffy, M. J., Serum tumor markers in breast cancer: are they of clinical value? Clinical Chemistry 2006, 52 (3), 345-51.
14.       Song, L.; Hanlon, D. W.; Chang, L.; Provuncher, G. K.; Kan, C. W.; Campbell, T. G.; Fournier, D. R.; Ferrell, E. P.; Rivnak, A. J.; Pink, B. A.; Minnehan, K. A.; Patel, P. P.; Wilson, D. H.; Till, M. A.; Faubion, W. A.; Duffy, D. C., Single molecule measurements of tumor necrosis factor alpha and interleukin-6 in the plasma of patients with Crohn's disease. J Immunol Methods 2011, 372 (1-2), 177-86.
15.       Harris, L.; Fritsche, H.; Mennel, R.; Norton, L.; Ravdin, P.; Taube, S.; Somerfield, M. R.; Hayes, D. F.; Bast, R. C., American Society of Clinical Oncology 2007 Update of Recommendations for the Use of Tumor Markers in Breast Cancer. Journal of Clinical Oncology 2007, 25 (33), 5287-5312.
16.       Anderson, K. S.; Ramachandran, N.; Wong, J.; Raphael, J. V.; Hainsworth, E.; Demirkan, G.; Cramer, D.; Aronzon, D.; Hodi, F. S.; Harris, L.; Logvinenko, T.; LaBaer, J., Application of Protein Microarrays for Multiplexed Detection of Antibodies to Tumor Antigens in Breast Cancer. Journal of Proteome Research 2008, 7 (4), 1490-1499.
17.       Wilson, D. H.; Hanlon, D. W.; Provuncher, G. K.; Chang, L.; Song, L.; Patel, P. P.; Ferrell, E. P.; Lepor, H.; Partin, A. W.; Chan, D. W.; Sokoll, L. J.; Cheli, C. D.; Thiel, R. P.; Fournier, D. R.; Duffy, D. C., Fifth-generation digital immunoassay for prostate-specific antigen by single molecule array technology. Clinical Chemistry 2011, 57 (12), 1712-21.
18.       Song, L.; Hanlon, D. W.; Chang, L.; Provuncher, G. K.; Kan, C. W.; Campbell, T. G.; Fournier, D. R.; Ferrell, E. P.; Rivnak, A. J.; Pink, B. A.; Minnehan, K. A.; Patel, P. P.; Wilson, D. H.; Till, M. A.; Faubion, W. A.; Duffy, D. C., Single molecule measurements of tumor necrosis factor α and interleukin-6 in the plasma of patients with Crohn's disease. Journal of Immunological Methods 2011, 372 (1–2), 177-186.
19.       Esquela-Kerscher, A.; Slack, F. J., Oncomirs [mdash] microRNAs with a role in cancer. Nat Rev Cancer 2006, 6 (4), 259-269.
20.       Lu, J.; Getz, G.; Miska, E. A.; Alvarez-Saavedra, E.; Lamb, J.; Peck, D.; Sweet-Cordero, A.; Ebert, B. L.; Mak, R. H.; Ferrando, A. A.; Downing, J. R.; Jacks, T.; Horvitz, H. R.; Golub, T. R., MicroRNA expression profiles classify human cancers. Nature 2005, 435 (7043), 834-838.
21.       Blenkiron, C.; Goldstein, L. D.; Thorne, N. P.; Spiteri, I.; Chin, S.-F. F.; Dunning, M. J.; Barbosa-Morais, N. L.; Teschendorff, A. E.; Green, A. R.; Ellis, I. O.; Tavaré, S.; Caldas, C.; Miska, E. A., MicroRNA expression profiling of human breast cancer identifies new markers of tumor subtype. Genome Biology 2007, 8 (10), R214+-R214+.
22.       Jain, P. K.; Shah, S.; Friedman, S. H., Patterning of Gene Expression Using New Photolabile Groups Applied to Light Activated RNAi. Journal of the American Chemical Society 2010, 133 (3), 440-446.
23.       Jørgensen, K. J.; Gøtzsche, P. C., Overdiagnosis in publicly organised mammography screening programmes: systematic review of incidence trends. BMJ 2009, 339.