In vivo tracking of individual stem cells labeled with nanowire lasers using multimodality imaging.

Emerging cell-based regenerative medicine and stem cell therapies have drawn wide attention in medical research and clinical practice to treat tissue damage and numerous incurable diseases. In vivo observation of the distribution, migration, and development of the transplanted cells is important for both understanding the mechanism and evaluating the treatment efficacy and safety. However, tracking the 3D migration trajectories for individual therapeutic cells in clinically relevant pathological environments remains technically challenging. Using a laser photocoagulation model in living rabbit eyes, this study demonstrates a multimodality imaging technology integrating optical coherence tomography (OCT), fluorescence microscopy (FM), and lasing emission for in vivo longitudinal tracking of the 3D migration trajectories of individual human retinal pigment epithelium cells (ARPE-19) labeled with CdS nanowires. With unique lasing spectra generated from the subtle microcavity differences, the surface-modified nanowires perform as distinct spectral identifiers for labeling individual ARPE-19 cells. Meanwhile, with strong optical scattering and natural fluorescence emission, CdS nanowires also served as OCT and FM contrast agents to indicate the spatial locations of the transplanted ARPE-19 cells. A longitudinal study of tracking individual ARPE-19 cells in rabbit eyes over a duration of 28 days was accomplished. This method could potentially promote an understanding of the pharmacodynamics and pharmacokinetics of implanted cells in the development of cell-based therapies.

Rapid and Quantitative In Vitro Evaluation of SARS-CoV-2 Neutralizing Antibodies and Nanobodies.

Neutralizing monoclonal antibodies and nanobodies have shown promising results as potential therapeutic agents for COVID-19. Identifying such antibodies and nanobodies requires evaluating the neutralization activity of a large number of lead molecules via biological assays, such as the virus neutralization test (VNT). These assays are typically time-consuming and demanding on-lab facilities. Here, we present a rapid and quantitative assay that evaluates the neutralizing efficacy of an antibody or nanobody within 1.5 h, does not require BSL-2 facilities, and consumes only 8 µL of a low concentration (ng/mL) sample for each assay run. We tested the human angiotensin-converting enzyme 2 (ACE2) binding inhibition efficacy of seven antibodies and eight nanobodies and verified that the IC50 values of our assay are comparable with those from SARS-CoV-2 pseudovirus neutralization tests. We also found that our assay could evaluate the neutralizing efficacy against three widespread SARS-CoV-2 variants. We observed increased affinity of these variants for ACE2, including the ß and γ variants. Finally, we demonstrated that our assay enables the rapid identification of an immune-evasive mutation of the SARS-CoV-2 spike protein, utilizing a set of nanobodies with known binding epitopes.

Bioresponsive microlasers with tunable lasing wavelength.

Lasing particles are emerging tools for amplifying light-matter interactions at the biointerface by exploiting its strong intensity and miniaturized size. Recent advances in implementing laser particles into living cells and tissues have opened a new frontier in biological imaging, monitoring, and tracking. Despite remarkable progress in micro- and nanolasers, lasing particles with surface functionality remain challenging due to the low mode-volume while maintaining a high Q-factor. Herein, we report the novel concept of bioresponsive microlasers by exploiting interfacial energy transfer based on whispering-gallery-mode (WGM) microdroplet cavities. Lasing wavelengths were manipulated by energy transfer-induced changes of a gain spectrum resulting from the binding molecular concentrations at the cavity surface. Both protein-based and enzymatic-based interactions were demonstrated, shedding light on the development of functional microlasers. Finally, tunable lasing wavelengths over a broad spectral range were achieved by selecting different donor/acceptor pairs. This study not only opens new avenues for biodetection, but also provides deep insights into how molecules modulate laser light at the biointerface, laying the foundation for the development of smart bio-photonic devices at the molecular level.

Optical coherence tomography and fluorescence microscopy dual-modality imaging for in vivo single-cell tracking with nanowire lasers.

Emerging cell-based therapies such as stem cell therapy and immunotherapy have attracted broad attention in both biological research and clinical practice. However, a long-standing technical gap of cell-based therapies is the difficulty of directly assessing treatment efficacy via tracking therapeutically administered cells. Therefore, imaging techniques to follow the in vivo distribution and migration of cells are greatly needed. Optical coherence tomography (OCT) is a clinically available imaging technology with ultrahigh-resolution and excellent imaging depth. It also shows great potential for in vivo cellular imaging. However, due to the homogeneity of current OCT cell labeling contrast agents (such as gold and polymer nanoparticles), only the distribution of entire cell populations can be observed. Precise tracking of the trajectory of individual single cells is not possible with such conventional contrast agents. Microlasers may provide a route to track unique cell identifiers given their small size, high emission intensities, rich emission spectra, and narrow linewidths. Here, we demonstrate that nanowire lasers internalized by cells provide both OCT and fluorescence signal. In addition, cells can be individually identified by the unique lasing emission spectra of the nanowires that they carry. Furthermore, single cell migration trajectories can be monitored both in vitro and in vivo with OCT and fluorescence microscopy dual-modality imaging system. Our study demonstrates the feasibility of nanowire lasers combined with the dual-modality imaging system for in vivo single cell tracking with a high spatial resolution and identity verification, an approach with great utility for stem cell and immunomodulatory therapies.

Rapid and quantitative detection of SARS-CoV-2 specific IgG for convalescent serum evaluation.

Convalescent serum with a high abundance of neutralization IgG is a promising therapeutic agent for rescuing COVID-19 patients in the critical stage. Knowing the concentration of SARS-CoV-2 S1-specific IgG is crucial in selecting appropriate convalescent serum donors. Here, we present a portable microfluidic ELISA technology for rapid (15 min), quantitative, and sensitive detection of anti-SARS-CoV-2 S1 IgG in human serum with only 8 µL sample volume. We first identified a humanized monoclonal IgG that has a high binding affinity and a relatively high specificity towards SARS-CoV-2 S1 protein, which can subsequently serve as the calibration standard of anti-SARS-CoV-2 S1 IgG in serological analyses. We then measured the abundance of anti-SARS-CoV-2 S1 IgG in 16 convalescent COVID-19 patients. Due to the availability of the calibration standard and the large dynamic range of our assay, we were able to identify "qualified donors" for convalescent serum therapy with only one fixed dilution factor (200 ×). Finally, we demonstrated that our technology can sensitively detect SARS-CoV-2 antigens (S1 and N proteins) with pg/mL level sensitivities in 40 min. Overall, our technology can greatly facilitate rapid, sensitive, and quantitative analysis of COVID-19 related markers for therapeutic, diagnostic, epidemiologic, and prognostic purposes.

Multiparameter urine analysis for quantitative bladder cancer surveillance of orthotopic xenografted mice.

The human-derived orthotopic xenograft mouse model is an effective platform for performing in vivo bladder cancer studies to examine tumor development, metastasis, and therapeutic effects of drugs. To date, the surveillance of tumor progression in real time for orthotopic bladder xenografts is highly dependent on semi-quantitative in vivo imaging technologies such as bioluminescence. While these imaging technologies can estimate tumor progression, they are burdened with requirements such as anesthetics, specialized equipment, and genetic modification of the injected cell line. Thus, a convenient and non-invasive technology to quantitatively monitor the growth of bladder cancer in orthotopic xenografts is highly desired. In this work, using a microfluidic chemiluminescent ELISA platform, we have successfully developed a rapid, multiparameter urine-based and non-invasive biomolecular prognostic technology for orthotopic bladder cancer xenografts. This method consists of two steps. First, the concentrations of a panel of four urinary biomarkers are quantified from the urine of mice bearing orthotopic bladder xenografts. Second, machine learning and principal component analysis (PCA) algorithms are applied to analyze the urinary biomarkers, and subsequently, a score is assigned to indicate the tumor growth. With this methodology, we have quantitatively monitored the orthotopic growth of human bladder cancer that was inoculated with low, medium, and high cancer cell numbers. We also employed this method and performed a proof of principle experiment to examine the in vivo therapeutic efficacy of the EGFR inhibitor, dacomitinib.

Rapid and sensitive detection of drugs of abuse in sweat by multiplexed capillary based immuno-biosensors.

Rapid and sensitive detection of drugs of abuse plays an important role in monitoring of drug use and treatment compliance. Sweat based drug analysis shows great advantages due to its non-invasive nature. However, most of the related methods developed to date are qualitative, slow, or costly, which significantly hinders their application in field use. Here we report rapid, sensitive, quantitative detection of drugs of abuse in sweat based on capillary arrays combined with competitive enzyme-linked immunosorbent assay. Using four common drugs of abuse, methadone, methamphetamine, amphetamine, and tetrahydrocannabinol, spiked in artificial sweat as a model system, we demonstrate rapid, quantitative, and multiplexed detection of the four drugs in ∼16 minutes with a low sweat volume (∼4 µL per analyte) and a large dynamic range (methadone: 0.0016 ng mL-1-1 ng mL-1; METH: 0.016 ng mL-1-25 ng mL-1; amphetamine: 0.005 ng mL-1-10 ng mL-1; THC: 0.02 ng mL-1-1000 ng mL-1). In addition, we show that the detection range can be tuned for different applications by adjusting the competitors' concentrations. Our work paves a way to develop an autonomous, portable, and cost-effective device for hospital testing, workplace drug-use screening, roadside testing, and patient monitoring in drug rehabilitation centers.

Fast and Reproducible ELISA Laser Platform for Ultrasensitive Protein Quantification.

Optofluidic lasers are currently of high interest for sensitive intracavity biochemical analysis. In comparison with conventional methods such as fluorescence and colorimetric detection, optofluidic lasers provide a method for amplifying small concentration differences in the gain medium, thus achieving high sensitivity. Here, we report the development of an on-chip ELISA (enzyme-linked immunosorbent assay) laser platform that is able to complete an assay in a short amount of time with small sample/reagent volumes, large dynamic range, and high sensitivity. The arrayed microscale reaction wells in the ELISA lasers can be microfabricated directly on dielectric mirrors, thus significantly improving the quality of the reaction wells and detection reproducibility. The details of the fabrication and characterization of those reaction wells on the mirror are described and the ELISA laser assay protocols are developed. Finally, we applied the ELISA laser to detecting IL-6, showing that a detection limit of about 0.1 pg/mL can be achieved in 1.5 h with 15 µL of sample/reagents per well. This work pushes the ELISA laser a step closer to solving problems in real-world biochemical analysis.

Turbidimetric inhibition immunoassay revisited to enhance its sensitivity via an optofluidic laser.

Turbidimetric inhibition immunoassay (TIIA) is a classic immunodiagnostic method that has been extensively used for biomarker detection. However, the low sensitivity of this technique hinders its applications in the early diagnosis of diseases. Here, a new concept, optofluidic laser TIIA (OFL-TIIA), is proposed and demonstrated for sensitive protein detection. In contrast to the immunoreaction in traditional TIIA, in which the single-pass laser loss is detected, the immunoreaction in the OFL-TIIA method takes place in a laser cavity, which considerably increases the loss induced by antigen-antibody complexes (AACs) via the amplification effect of the laser. A commercial IgG TIIA kit was selected as a demonstrative model to characterize the performance of OFL-TIIA. A wide dynamic range of five orders of magnitude with an exceptional limit of detection (LOD) (1.8 × 10-10 g/L) was achieved. OFL-TIIA is a fast, sensitive, and low-cost immunoassay with a simple homogeneous and wash-free process and low-volume sample consumption, thus providing a new detection platform for disease diagnostics.

Rapid Mouse Follicle Stimulating Hormone Quantification and Estrus Cycle Analysis Using an Automated Microfluidic Chemiluminescent ELISA System.

Follicle stimulating hormone (FSH) plays a critical role in female reproductive development and homeostasis. The blood/serum concentration of FSH is an important marker for reporting multiple endocrinal functions. The standardized method for mouse FSH (mFSH) quantification based on radioimmunoassay (RIA) suffers from long assay time (∼2 days), relatively low sensitivity, larger sample volume (60 µL), and small dynamic range (2-60 ng/mL); thus, it is insufficient for monitoring fast developing events with relatively small mFSH fluctuations (e.g., estrous cycles of mammals). Here, we developed an automated microfluidic chemiluminescent ELISA device along with the disposal sensor array and the corresponding detection protocol for rapid and quantitative analysis of mFSH from mouse tail serum samples. With this technology, highly sensitive quantification of mFSH can be accomplished within 30 min using only 8 µL of the serum sample. It is further shown that our technique is able to generate results comparable to RIA but has a significantly improved dynamic range that covers 0.5-250 ng/mL. The performance of this technology was evaluated with blood samples collected from ovariectomized animals and animals with reimplanted ovarian tissues, which restored ovarian endocrine function and correlated with estrus cycle analysis study.

Distributed fibre optofluidic laser for chip-scale arrayed biochemical sensing.

Optofluidic lasers (OFLs) are an emerging technological platform for biochemical sensing, and their good performance especially high sensitivity has been demonstrated. However, high-throughput detection with an OFL remains a major challenge due to the lack of reproducible optical microcavities. Here, we introduce the concept of a distributed fibre optofluidic laser (DFOFL) and demonstrate its potential for high-throughput sensing applications. Due to the precise fibre geometry control via fibre drawing, a series of identical optical microcavities uniformly distributed along a hollow optical fibre (HOF) can be achieved to obtain a one-dimensional (1D) DFOFL. An enzymatic reaction catalysed by horseradish peroxidase (HRP) can be monitored over time, and the HRP concentration is detected by DFOFL-based arrayed colorimetric detection. Experimentally, five-channel detection in parallel with imaging has been demonstrated. Theoretically, spatial multiplexing of hundreds of channels is achievable with DFOFL-based detection. The DFOFL wavelength is tuned over hundreds of nanometers by optimizing the dye concentration or reconfiguring the liquid gain materials. Extending this concept to a two-dimensional (2D) chip through wavelength multiplexing can further enhance its multi-functionality, including multi-sample detection and spectral analysis. This work opens the door to high-throughput biochemical sensing.

A robust tissue laser platform for analysis of formalin-fixed paraffin-embedded biopsies.

Laser emission-based detection and imaging technology has attracted significant interest in biomedical research due to its high sensitivity, narrow linewidth, and superior spectral and spatial resolution. Recent advances have further revealed the potential to use laser emission to investigate chromatin dynamics, as well as to diagnose cancer tissues based on nuclear biomarkers. To move the laser emission based detection technology a step further towards practical use, in this work, we developed a highly robust tissue laser platform by microfabricating an SU8 spacer with a fixed height on the top mirror of the Fabry-Pérot (FP) cavity, which allows generation of reproducible and stable lasing results regardless of tissue thickness. Then we applied this platform to achieve lasing emission from formalin-fixed, paraffin-embedded (FFPE) lung tissues, which account for an overwhelming fraction of tissues collected for research and clinical use worldwide. We further showed that the cancer and normal FFPE lung tissues can be distinguished by their respective lasing thresholds. Two different tissue thicknesses (10 µm and 5 µm) commonly used in pathological labs were explored. Finally, we tested three additional types of tissues (colon, stomach, and breast) that were prepared independently by lab technicians in a pathology lab in China and shipped to the US in order to validate the general applicability and practicality of the laser emission-based technology as well as the corresponding sample preparation protocol and the tissue laser platform. Our work will not only vastly broaden the applications of laser emission-based detection/imaging technology but also help translate it from the laboratory to an automated system for clinical practice that may eventually benefit biomedicine and biological research.

Laser-emission imaging of nuclear biomarkers for high-contrast cancer screening and immunodiagnosis.

Detection of nuclear biomarkers such as nucleic acids and nuclear proteins is critical for early-stage cancer diagnosis and prognosis. Conventional methods relying on morphological assessment of cell nuclei in histopathology slides may be subjective, whereas colorimetric immunohistochemical and fluorescence-based imaging are limited by strong light absorption, broad-emission bands and low contrast. Here, we describe the development and use of a scanning laser-emission-based microscope that maps lasing emissions from nuclear biomarkers in human tissues. 41 tissue samples from 35 patients labelled with site-specific and biomarker-specific antibody-conjugated dyes were sandwiched in a Fabry-Pérot microcavity while an excitation laser beam built a laser-emission image. We observed multiple sub-cellular lasing emissions from cancer cell nuclei, with a threshold of tens of µJ/mm2, sub-micron resolution (<700 nm), and a lasing band in the few-nanometre range. Different lasing thresholds of nuclei in cancer and normal tissues enabled the identification and multiplexed detection of nuclear proteomic biomarkers, with a high sensitivity for early-stage cancer diagnosis. Laser-emission-based cancer screening and immunodiagnosis might find use in precision medicine and facilitate research in cell biology.

Sensitive sulfide ion detection by optofluidic catalytic laser using horseradish peroxidase (HRP) enzyme.

We report an optofluidic catalytic laser for sensitive sulfide ion detection. In the catalytic reaction, horseradish peroxidase (HRP) enzyme is used for catalyzing the non-fluorescent substrate, 10-Acetyl-3,7-dihydroxyphenox-azine (ADHP), to produce highly fluorescent resorufin, which was used as gain medium for lasing. Using sulfide ions as inhibitors, the catalytic reaction slows down, resulting in a delay in the lasing onset time, which is used as the sensing signal. The sensing mechanism of the catalytic laser is theoretically analyzed and the performance is experimentally characterized. Sulfide anion is chosen as a model ion because of its broad adverse impacts on both environment and human health. Due to the optical feedback provided by the laser, the small difference in the sulfide ion concentration can be amplified. Consequently, a detection limit of 10nM is achieved with a dynamic range as large as three orders of magnitude, representing significant improvement over the traditional fluorescence and colorimetric methods. This work will open a door to a new catalytic-laser-based chemical sensing platform for detecting a wide range of species that could inhibit the catalytic reaction.

Glass capillary based microfluidic ELISA for rapid diagnostics.

Enzyme-linked immunosorbent assay (ELISA) is widely used in medical diagnostics and fundamental biological research due to its high specificity and reproducibility. However, the traditional 96-well-plate based ELISA still suffers from several notable drawbacks, such as long assay time (4-6 hours), burdensome procedures and large sample/reagent volumes (∼100 µl), which significantly limit traditional ELISA's applications in rapid clinical diagnosis and quasi-real-time prognosis of some fast-developing diseases. Here, we developed a user friendly glass capillary array based microfluidic ELISA device. Benefiting from the high surface-to-volume ratio of the capillary and the rapid chemiluminescent photo-imaging method with a commercial camera, our capillary based ELISA device significantly reduced the sample volume to 20 µL and shortened the total assay time to as short as 16 minutes (including detection time), which represent approximately 10-fold and 5-fold reduction in assay time and sample volume, respectively, in comparison with the traditional plate-based method. Furthermore, through the double exposure method, a nearly 10-fold increase in the detection dynamic range was achieved over the traditional well-based ELISA. Our device can be broadly used in rapid biochemical analysis for biomedicine and research/development laboratories.

The inhibitory effect of IFN-γ on protease HTRA1 expression in rheumatoid arthritis.

The high temperature requirement A1 (HTRA1) is a potent protease involved in many diseases, including rheumatoid arthritis (RA). However, the regulatory mechanisms that control HTRA1 expression need to be determined. In this study, we demonstrated that IFN-γ significantly inhibited the basal and LPS-induced HTRA1 expression in fibroblasts and macrophages, which are two major cells for HTRA1 production in RA. Importantly, the inhibitory effect of IFN-γ on HTRA1 expression was evidenced in collagen-induced arthritis (CIA) mouse models and in human RA synovial cells. In parallel with the enhanced CIA incidence and pathological changes in IFN-γ-deficient mice, HTRA1 expression in the joint tissues was also increased as determined by real-time PCR and Western blots. IFN-γ deficiency increased the incidence of CIA and the pathological severity in mice. Neutralization of HTRA1 by Ab significantly reversed the enhanced CIA frequency and severity in IFN-γ-deficient mice. Mechanistically, IFN-γ negatively controls HTRA1 expression through activation of p38 MAPK/STAT1 pathway. Dual luciferase reporter assay and chromatin immunoprecipitation analysis showed that STAT1 could directly bind to HTRA1 promoter after IFN-γ stimulation. This study offers new insights into the molecular regulation of HTRA1 expression and its role in RA pathogenesis, which may have significant impact on clinical therapy for RA and possibly other HTRA1-related diseases, including osteoarthritis, age-related macular degeneration, and cancer.