Pierre N. Floriano

A Microchip CD4 Counting Method for HIV Monitoring in Resource-Poor Settings

Background More than 35 million people in developing countries are living with HIV infection. An enormous global effort is now underway to bring antiretroviral treatment to at least 3 million of those infected. While drug prices have dropped considerably, the cost and technical complexity of laboratory tests essential for the management of HIV disease, such as CD4 cell counts, remain prohibitive. New, simple, and affordable methods for measuring CD4 cells that can be implemented in resource-scarce settings are urgently needed. Methods and Findings Here we describe the development of a prototype for a simple, rapid, and affordable method for counting CD4 lymphocytes. Microliter volumes of blood without further sample preparation are stained with fluorescent antibodies, captured on a membrane within a miniaturized flow cell and imaged through microscope optics with the type of charge-coupled device developed for digital camera technology. An associated computer algorithm converts the raw digital image into absolute CD4 counts and CD4 percentages in real time. The accuracy of this prototype system was validated through testing in the United States and Botswana, and showed close agreement with standard flow cytometry (r = 0.95) over a range of absolute CD4 counts, and the ability to discriminate clinically relevant CD4 count thresholds with high sensitivity and specificity. Conclusion Advances in the adaptation of new technologies to biomedical detection systems, such as the one described here, promise to make complex diagnostics for HIV and other infectious diseases a practical global reality.

Nano-bio-chips for high performance multiplexed protein detection: Determinations of cancer biomarkers in serum and saliva using quantum dot bioconjugate labels

The integration of semiconductor nanoparticle quantum dots (QDs) into a modular, microfluidic biosensor for the multiplexed quantitation of three important cancer markers, carcinoembryonic antigen (CEA), cancer antigen 125 (CA125), and Her-2/Neu (C-erbB-2) was achieved. The functionality of the integrated sample processing, analyte capture and detection modalities was demonstrated using both serum and whole saliva specimens. Here, nano-bio-chips that employed a fluorescence transduction signal with QD-labeled detecting antibody were used in combination with antigen capture by a microporous agarose bead array supported within a microfluidics ensemble so as to complete the sandwich-type immunoassay. The utilization of QD probes in this miniaturized biosensor format resulted in signal amplification 30 times relative to that of standard molecular fluorophores as well as affording a reduction in observed limits of detection by nearly 2 orders of magnitude (0.02 ng/mL CEA; 0.11 pM CEA) relative to enzyme-linked immunosorbent assay (ELISA). Assay validation studies indicate that measurements by the nano-bio-chip system correlate to standard methods at R(2)=0.94 and R(2)=0.95 for saliva and serum, respectively. This integrated nano-bio-chip assay system, in tandem with next-generation fluorophores, promises to be a sensitive, multiplexed tool for important diagnostic and prognostic applications.

Use of saliva-based nano-biochip tests for acute myocardial infarction at the point of care: A feasibility study

BACKGROUND For adults with chest pain, the electrocardiogram (ECG) and measures of serum biomarkers are used to screen and diagnose myocardial necrosis. These measurements require time that can delay therapy and affect prognosis. Our objective was to investigate the feasibility and utility of saliva as an alternative diagnostic fluid for identifying biomarkers of acute myocardial infarction (AMI). METHODS We used Luminex and lab-on-a-chip methods to assay 21 proteins in serum and unstimulated whole saliva procured from 41 AMI patients within 48 h of chest pain onset and from 43 apparently healthy controls. Data were analyzed by use of logistic regression and area under curve (AUC) for ROC analysis to evaluate the diagnostic utility of each biomarker, or combinations of biomarkers, in screening for AMI. RESULTS Both established and novel cardiac biomarkers demonstrated significant differences in concentrations between patients with AMI and controls without AMI. The saliva-based biomarker panel of C-reactive protein, myoglobin, and myeloperoxidase exhibited significant diagnostic capability (AUC = 0.85, P < 0.0001) and in conjunction with ECG yielded strong screening capacity for AMI (AUC = 0.96) comparable to that of the panel (brain natriuretic peptide, troponin-I, creatine kinase-MB, myoglobin; AUC = 0.98) and far exceeded the screening capacity of ECG alone (AUC approximately 0.6). En route to translating these findings to clinical practice, we adapted these unstimulated whole saliva tests to a novel lab-on-a-chip platform for proof-of-principle screens for AMI. CONCLUSIONS Complementary to ECG, saliva-based tests within lab-on-a-chip systems may provide a convenient and rapid screening method for cardiac events in prehospital stages for AMI patients.

Lab‐on‐a‐Chip Methods for Point‐of‐Care Measurements of Salivary Biomarkers of Periodontitis

Abstract:  Salivary secretions contain a variety of molecules that reflect important pathophysiological activities. Quantitative changes of specific salivary biomarkers could have significance in the diagnosis and management of both oral and systemic diseases. Modern point‐of‐care technologies with enhanced detection capabilities are needed to implement a significant advancement in salivary diagnostics. One such promising technology is the recently described lab‐on‐a‐chip (LOC) assay system, in which assays are performed on chemically sensitized beads populated into etched silicon wafers with embedded fluid handling and optical detection capabilities. Using this LOC system, complex assays can be performed with small sample volumes, short analysis times, and markedly reduced reagent costs. This report describes the use of LOC methodologies to assess the levels of interleukin‐1β (IL‐1β), C‐reactive protein (CRP), and matrix metalloproteinase‐8 (MMP‐8) in whole saliva, and the potential use of these biomarkers for diagnosing and categorizing the severity and extent of periodontitis. This study demonstrates that the results achieved by the LOC approach are in agreement with those acquired with standard enzyme‐linked immunosorbent assay (ELISA), with significant IL‐1β and MMP‐8 elevations in whole saliva of periodontitis patients. Furthermore, because of the superior detection capacities associated with the LOC approach, unlike those with ELISA, significant differences in CRP levels between periodontitis patients and normal subjects are observed. Finally, principal component analysis (PCA) is performed to yield an efficient method to discriminate between periodontally healthy and unhealthy patients, thus increasing the diagnostic value of these biomarkers for periodontitis when examined with the integrated LOC sensor system.

Programmable nano-bio-chip sensors: Analytical meets clinical

There have been many recent advances in the nano-bio-chip analysis methodology with implications for a number of high-morbidity diseases including HIV, cancer, and heart disease. (To listen to a podcast about this article, please go to the Analytical Chemistry multimedia page at pubs.acs.org/page/ancham/audio/index.html .).

Integration of semiconductor quantum dots into nano-bio-chip systems for enumeration of CD4+ T cell counts at the point-of-need

Recent humanitarian efforts have led to the widespread release of antiretroviral drugs for the treatment of the more than 33 million HIV afflicted people living in resource-scarce settings. Here, the enumeration of CD4+ T lymphocytes is required to establish the level at which the immune system has been compromised. The gold standard method used in developed countries, based on flow cytometry, though widely accepted and accurate, is precluded from widespread use in resource-scarce settings due to its high expense, high technical requirements, difficulty in operation-maintenance and the lack of portability for these sophisticated laboratory-confined systems. As part of continuing efforts to develop practical diagnostic instrumentation, the integration of semiconductor nanocrystals (quantum dots, QDs) into a portable microfluidic-based lymphocyte capture and detection device is completed. This integrated system is capable of isolating and counting selected lymphocyte sub-populations (CD3+CD4+) from whole blood samples. By combining the unique optical properties of the QDs with the sample handling capabilities and cost effectiveness of novel microfluidic systems, a practical, portable lymphocyte measurement modality that correlates nicely with flow cytometry (R2 = 0.97) has been developed. This QD-based system reduces the optical requirements significantly relative to molecular fluorophores and the mini-CD4 counting device is projected to be suitable for use in both point-of-need and resource-scarce settings.

Nano-bio-chip sensor platform for examination of oral exfoliative cytology

Oral cancer is a deadly and disfiguring disease that could greatly benefit from new diagnostic approaches enabling early detection. In this pilot study, we describe a nano-bio-chip (NBC) sensor technique for analysis of oral cancer biomarkers in exfoliative cytology specimens, targeting both biochemical and morphologic changes associated with early oral tumorigenesis. Here, oral lesions from 41 dental patients, along with normal epithelium from 11 healthy volunteers, were sampled using a noninvasive brush biopsy technique. Specimens were enriched, immunolabeled, and imaged in the NBC sensor according to previously established assays for the epidermal growth factor receptor (EGFR) biomarker and cytomorphometry. A total of 51 measurement parameters were extracted using custom image analysis macros, including EGFR labeling intensity, cell and nuclear size, and the nuclear-to-cytoplasmic ratio. Four key parameters were significantly elevated in both dysplastic and malignant lesions relative to healthy oral epithelium, including the nuclear area and diameter (P < 0.0001), the nuclear-to-cytoplasmic ratio (P < 0.0001), and EGFR biomarker expression (P < 0.03). Further examination using logistic regression and receiver operating characteristic curve analyses identified morphologic features as the best predictors of disease (area under the curve ≤0.93) individually, whereas a combination of all features further enhanced discrimination of oral cancer and precancerous conditions (area under the curve, 0.94) with high sensitivity and specificity. Further clinical trials are necessary to validate the regression model and evaluate other potential biomarkers, but this pilot study supports the NBC sensor technique as a promising new diagnostic tool for early detection of oral cancer, which could enhance patient care and survival. Cancer Prev Res; 3(4); 518–28. ©2010 AACR.

Perspective on Diagnostics for Global Health

Diagnostic applications for global health have exploded in the past ten years. Numerous articles have been generated on global health priorities, constraints of resource-limited settings, and technological innovations for diagnostics development, including several comprehensive reviews [1]-[3]. In this article, we aim to provide 1) a focused summary of the most highly needed diagnostics, 2) a discussion of noteworthy recent developments of technologies in the field, and 3) a perspective on the evolution, challenges, and future directions of diagnostics for global health applications.

Utility of Salivary Biomarkers for Demonstrating Acute Myocardial Infarction

The comparative utility of serum and saliva as diagnostic fluids for identifying biomarkers of acute myocardial infarction (AMI) was investigated. The goal was to determine if salivary biomarkers could facilitate a screening diagnosis of AMI, especially in cases of non-ST elevation MI (NSTEMI), since these cases are not readily identified by electrocardiogram (ECG). Serum and unstimulated whole saliva (UWS) collected from 92 AMI patients within 48 hours of chest pain onset and 105 asymptomatic healthy control individuals were assayed for 13 proteins relevant to cardiovascular disease, by Beadlyte technology (Luminex®) and enzyme immunoassays. Data were analyzed with concentration cut-points, ECG findings, logistic regression (LR) (adjusted for matching for age, gender, race, smoking, number of teeth, and oral health status), and classification and regression tree (CART) analysis. A sensitivity analysis was conducted by repetition of the CART analysis in 58 cases and 58 controls, each matched by age and gender. Serum biomarkers demonstrated AMI sensitivity and specificity superior to that of saliva, as determined by LR and CART. The predominant discriminators in serum by LR were troponin I (TnI), B-type natriuretic peptide (BNP), and creatine kinase-MB (CK-MB), and TnI and BNP by CART. In saliva, LR identified C-reactive protein (CRP) as the biomarker most predictive of AMI. A combination of smoking tobacco, UWS CRP, CK-MB, sCD40 ligand, gender, and number of teeth identified AMI in the CART decision trees. When ECG findings, salivary biomarkers, and confounders were included, AMI was predicted with 80.0% sensitivity and 100% specificity. These analyses support the potential utility of salivary biomarker measurements used with ECG for the identification of AMI. Thus, saliva-based tests may provide additional diagnostic screening information in the clinical course for patients suspected of having an AMI.

Porous Bead-Based Diagnostic Platforms: Bridging the Gaps in Healthcare

Advances in lab-on-a-chip systems have strong potential for multiplexed detection of a wide range of analytes with reduced sample and reagent volume; lower costs and shorter analysis times. The completion of high-fidelity multiplexed and multiclass assays remains a challenge for the medical microdevice field; as it struggles to achieve and expand upon at the point-of-care the quality of results that are achieved now routinely in remote laboratory settings. This review article serves to explore for the first time the key intersection of multiplexed bead-based detection systems with integrated microfluidic structures alongside porous capture elements together with biomarker validation studies. These strategically important elements are evaluated here in the context of platform generation as suitable for near-patient testing. Essential issues related to the scalability of these modular sensor ensembles are explored as are attempts to move such multiplexed and multiclass platforms into large-scale clinical trials. Recent efforts in these bead sensors have shown advantages over planar microarrays in terms of their capacity to generate multiplexed test results with shorter analysis times. Through high surface-to-volume ratios and encoding capabilities; porous bead-based ensembles; when combined with microfluidic elements; allow for high-throughput testing for enzymatic assays; general chemistries; protein; antibody and oligonucleotide applications.