Nien Tsu Huang

Recent advancements in optofluidics-based single-cell analysis: optical on-chip cellular manipulation, treatment, and property detection.

Cellular analysis plays important roles in various biological applications, such as cell biology, drug development, and disease diagnosis. Conventional cellular analysis usually measures the average response from a whole cell group. However, bulk measurements may cause misleading interpretations due to cell heterogeneity. Another problem is that current cellular analysis may not be able to differentiate various subsets of cell populations, each exhibiting a different behavior than the others. Single-cell analysis techniques are developed to analyze cellular properties, conditions, or functional responses in a large cell population at the individual cell level. Integrating optics with microfluidic platforms provides a well-controlled microenvironment to precisely control single cell conditions and perform non-invasive high-throughput analysis. This paper reviews recent developments in optofluidic technologies for various optics-based single-cell analyses, which involve single cell manipulation, treatment, and property detection. Finally, we provide our views on the future development of integrated optics with microfluidics for single-cell analysis and discuss potential challenges and opportunities of this emerging research field in biological applications.

Integrated Nanoplasmonic Sensing for Cellular Functional Immunoanalysis Using Human Blood

Localized surface plasmon resonance (LSPR) nanoplasmonic effects allow for label-free, real-time detection of biomolecule binding events on a nanostructured metallic surface with simple optics and sensing tunability. Despite numerous reports on LSPR bionanosensing in the past, no study thus far has applied the technique for a cytokine secretion assay using clinically relevant immune cells from human blood. Cytokine secretion assays, a technique to quantify intercellular-signaling proteins secreted by blood immune cells, allow determination of the functional response of the donor’s immune cells, thus providing valuable information about the immune status of the donor. However, implementation of LSPR bionanosensing in cellular functional immunoanalysis based on a cytokine secretion assay poses major challenges primarily owing to its limited sensitivity and a lack of sufficient sample handling capability. In this paper, we have developed a label-free LSPR biosensing technique to detect cell-secreted tumor necrosis factor (TNF)-α cytokines in clinical blood samples. Our approach integrates LSPR bionanosensors in an optofluidic platform that permits trapping and stimulation of target immune cells in a microfluidic chamber with optical access for subsequent cytokine detection. The on-chip spatial confinement of the cells is the key to rapidly increasing a cytokine concentration high enough for detection by the LSPR setup, thereby allowing the assay time and sample volume to be significantly reduced. We have successfully applied this approach first to THP-1 cells and then later to CD45 cells isolated directly from human blood. Our LSPR optofluidics device allows for detection of TNF-α secreted from cells as few as 1000, which translates into a nearly 100 times decrease in sample volume than conventional cytokine secretion assay techniques require. We achieved cellular functional immunoanalysis with a minimal blood sample volume (3 μL) and a total assay time 3 times shorter than that of the conventional enzyme-linked immunosorbent assay (ELISA).

Emerging microfluidic tools for functional cellular immunophenotyping: a new potential paradigm for immune status characterization.

Rapid, accurate, and quantitative characterization of immune status of patients is of utmost importance for disease diagnosis and prognosis, evaluating efficacy of immunotherapeutics and tailoring drug treatments. Immune status of patients is often dynamic and patient-specific, and such complex heterogeneity has made accurate, real-time measurements of patient immune status challenging in the clinical setting. Recent advances in microfluidics have demonstrated promising applications of the technology for immune monitoring with minimum sample requirements and rapid functional immunophenotyping capability. This review will highlight recent developments of microfluidic platforms that can perform rapid and accurate cellular functional assays on patient immune cells. We will also discuss the future potential of integrated microfluidics to perform rapid, accurate, and sensitive cellular functional assays at a single-cell resolution on different types or subpopulations of immune cells, to provide an unprecedented level of information depth on the distribution of immune cell functionalities. We envision that such microfluidic immunophenotyping tools will allow for comprehensive and systems-level immunomonitoring, unlocking the potential to transform experimental clinical immunology into an information-rich science.

Optofluidic detection for cellular phenotyping

Quantitative analysis of the output of processes and molecular interactions within a single cell is highly critical to the advancement of accurate disease screening and personalized medicine. Optical detection is one of the most broadly adapted measurement methods in biological and clinical assays and serves cellular phenotyping. Recently, microfluidics has obtained increasing attention due to several advantages, such as small sample and reagent volumes, very high throughput, and accurate flow control in the spatial and temporal domains. Optofluidics, which is the attempt to integrate optics with microfluidics, shows great promise to enable on-chip phenotypic measurements with high precision, sensitivity, specificity, and simplicity. This paper reviews the most recent developments of optofluidic technologies for cellular phenotyping optical detection.

Label-free cytokine micro- and nano-biosensing towards personalized medicine of systemic inflammatory disorders

Systemic inflammatory disorders resulting from infection, trauma, surgery, and severe disease conditions pose serious threats to human health leading to organ dysfunction, organ failure, and mortality. The highly complex and dynamic nature of the immune system experiencing acute inflammation makes immunomodulatory therapy blocking pro-inflammatory cytokines very challenging. Successful therapy requires the ability to determine appropriate anti-cytokine drugs to be delivered at a right dose in a timely manner. Label-free micro- and nano-biosensors hold the potential to overcome the current challenges, enabling cytokine-targeted treatments to be tailored according to the immune status of an individual host with their unique cytokine biomarker detection capabilities. This review studies the recent progress in label-free cytokine biosensors, summarizes their performances and potential merits, and discusses future directions for their advancements to meet challenges towards personalized anti-cytokine drug delivery.

Aqueous two-phase systems enable multiplexing of homogeneous immunoassays

Quantitative measurement of protein biomarkers is critical for biomarker validation and early disease detection. Current multiplex immunoassays are time consuming costly and can suffer from low accuracy. For example, multiplex ELISAs require multiple, tedious, washing and blocking steps. Moreover, they suffer from nonspecific antibody cross-reactions, leading to high background and false-positive signals. Here, we show that co-localizing antibody-bead pairs in an aqueous two-phase system (ATPS) enables multiplexing of sensitive, no-wash, homogeneous assays, while preventing nonspecific antibody cross-reactions. Our cross-reaction-free, multiplex assay can simultaneously detect picomolar concentrations of four protein biomarkers ((C-X-C motif) ligand 10 (CXCL10), CXCL9, interleukin (IL)-8 and IL-6) in cell supernatants using a single assay well. The potential clinical utility of the assay is demonstrated by detecting diagnostic biomarkers (CXCL10 and CXCL9) in plasma from 88 patients at the onset of the clinical symptoms of chronic graft-versus-host disease (GVHD).

Multiplexed spectral signature detection for microfluidic color-coded bioparticle flow.

Here, we report a high-speed photospectral detection technique capable of discriminating subtle variations of spectral signature among fluorescently labeled cells and microspheres flowing in a microfluidic channel. The key component used in our study is a strain-tunable nanoimprinted grating microdevice coupled with a photomultiplier tube (PMT). The microdevice permits acquisition of the continuous spectral profiles of multiple fluorescent emission sources at 1 kHz. Optically connected to a microfluidic flow chamber via a multimode optical fiber, our multiwavelength detection platform allows for cytometric measurement of cell groups emitting nearly identical fluorescence signals with a maximum emission wavelength difference as small as 5 nm. The same platform also allows us to demonstrate microfluidic flow cytometry of four different microsphere types in a wavelength bandwidth as narrow as 40 nm at a high (>85%) confidence level. Our study shows that detection of fluorescent spectral signatures at high speed and high resolution can expand specificity of multicolor flow cytometry. The enhanced capability enables multiplexed analysis of color-coded bioparticles based on single-laser excitation and single-detector spectroscopy in a microfluidic setting. The fluorescence signal discrimination power achieved by the optofluidic technology holds great promise to enable quantification of cellular parameters with higher accuracy as well as enumeration of a larger number of cell types than conventional flow cytometric methods.

High-speed tuning of visible laser wavelength using a nanoimprinted grating optical tunable filter

We report on a microelectromechanical tunable optical filter incorporating strain-tunable nanoimprinted elastomeric grating with a pitch varied by 18%. This device enables tuning of optical fiber-guided laser wavelength between lambda=473 and 532 nm within 0.5 ms by mechanically modulating the pitch with a silicon microactuator. We also demonstrate the use of the device for obtaining two-color images of livedead-stained cells with the color intensity ratio varied by the actuator voltage applied. The small structure of the device integrated on a silicon chip may be used in portable systems for optical switching and spectroscopy.

Pre-Clinical Tests of an Integrated CMOS Biomolecular Sensor for Cardiac Diseases Diagnosis

Coronary artery disease and its related complications pose great threats to human health. In this work, we aim to clinically evaluate a CMOS field-effect biomolecular sensor for cardiac biomarkers, cardiac-specific troponin-I (cTnI), N-terminal prohormone brain natriuretic peptide (NT-proBNP), and interleukin-6 (IL-6). The CMOS biosensor is implemented via a standard commercialized 0.35 μm CMOS process. To validate the sensing characteristics, in buffer conditions, the developed CMOS biosensor has identified the detection limits of IL-6, cTnI, and NT-proBNP as being 45 pM, 32 pM, and 32 pM, respectively. In clinical serum conditions, furthermore, the developed CMOS biosensor performs a good correlation with an enzyme-linked immuno-sorbent assay (ELISA) obtained from a hospital central laboratory. Based on this work, the CMOS field-effect biosensor poses good potential for accomplishing the needs of a point-of-care testing (POCT) system for heart disease diagnosis.

Capturing CD4 cells using a functionalized circular microfluidic device and glutaraldehyde as biolinker for tuberculosis detection and diagnosis

It is estimated that about one-third of the world’s population has already been infected by tuberculosis. Mycobacterium tuberculosis, in general, can result in an active case of tuberculosis in approximately 5%-10% of those who suffer from latent tuberculosis and the chance of becoming ill is the highest within one of year of getting the disease. Although a newly developed methods called interferon gamma release assay (IGRA) can monitor CD4 cells secreted cytokine to diagnose tuberculosis (TB) condition. However, it is difficult to count total numbers of cytokine secreted CD4 cells, which make the diagnosis less accurate. Therefore, we develop a functionalized polydimethylsiloxane (PDMS) device using glutaraldehyde to capture CD4 cells. To enhance the capture efficiency, we use COMSOL simulation to optimize the arrangement of PDMS micro pillars to make cells uniformly distributed in the device. Our preliminary data showed the microfluidic configuration in a circular shape with HCP patterned micro pillars turned 30 degrees offers the highest cell capture rate.