Bo Ram Oh

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).

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.

Multiple MoS2 Transistors for Sensing Molecule Interaction Kinetics.

Atomically layered transition metal dichalcogenides (TMDCs) exhibit a significant potential to enable next-generation low-cost transistor biosensors that permit single-molecule-level quantification of biomolecules. To realize such potential biosensing capability, device-oriented research is needed for calibrating the sensor responses to enable the quantification of the affinities/kinetics of biomolecule interactions. In this work, we demonstrated MoS2-based transistor biosensors capable of detecting tumor necrosis factor – alpha (TNF-α) with a detection limit as low as 60 fM. Such a detection limit was achieved in both linear and subthreshold regimes of MoS2 transistors. In both regimes, all sets of transistors exhibited consistent calibrated responses with respect to TNF-α concentration, and they resulted in a standard curve, from which the equilibrium constant of the antibody-(TNF-α) pair was extracted to be KD = 369 ± 48 fM. Based on this calibrated sensor model, the time-dependent binding kinetics was also measured and the association/dissociation rates of the antibody-(TNF-α) pair were extracted to be (5.03 ± 0.16) × 108 M−1s−1 and (1.97 ± 0.08) × 10−4 s−1, respectively. This work advanced the critical device physics for leveraging the excellent electronic/structural properties of TMDCs in biosensing applications as well as the research capability in analyzing the biomolecule interactions with fM-level sensitivities.

Fabrication and comparison of MoS2 and WSe2 field-effect transistor biosensors

The authors present a study on the evolution behaviors of the transfer characteristics of MoS2 and WSe2 field-effect transistor biosensors when they are subjected to tumor necrosis factor-alpha and streptavidin solutions with varying analyte concentrations. Both MoS2 and WSe2 sensors exhibit very low detection limits (∼60 fM for tumor necrosis factor-alpha detection; ∼70 fM for streptavidin detection). However, WSe2 sensors exhibit the higher linear-regime sensitivities in comparison with MoS2 sensors. In particular, WSe2 sensors exhibit high linear-regime sensitivities up to ∼1.54%/fM for detecting streptavidin at a concentration of ∼70 fM. Such relatively higher sensitivities obtained from WSe2 sensors are attributed to their intrinsic ambipolar transfer characteristics, which make their ON-state carrier concentrations significantly lower than those of MoS2 sensors, and therefore, the target-molecule-induced doping effect results in more prominent channel conductance modulation in WSe2 transistor sensor...

Two different device physics principles for operating MoS2 transistor biosensors with femtomolar-level detection limits

We experimentally identify two different physics principles for operating MoS2 transistor biosensors, which depend on antibody functionalization locations. If antibodies are functionalized on an insulating layer coated on a MoS2 transistor, antibody-antigen binding events mainly modify the transistor threshold voltage, which can be explained by the conventional capacitor model. If antibodies are directly grafted on the MoS2 transistor channel, the binding events mainly modulate the ON-state transconductance of the transistor, which is attributed to the antigen-induced disordered potential in the MoS2 channel. This work advances the device physics for simplifying the transistor biosensor structures targeting for femtomolar-level quantification of biomolecules.

Biotunable Nanoplasmonic Filter on Few-Layer MoS2 for Rapid and Highly Sensitive Cytokine Optoelectronic Immunosensing

Monitoring of the time-varying immune status of a diseased host often requires rapid and sensitive detection of cytokines. Metallic nanoparticle-based localized surface plasmon resonance (LSPR) biosensors hold promise to meet this clinical need by permitting label-free detection of target biomolecules. These biosensors, however, continue to suffer from relatively low sensitivity as compared to conventional immunoassay methods that involve labeling processes. Their response speeds also need to be further improved to enable rapid cytokine quantification for critical care in a timely manner. In this paper, we report an immunobiosensing device integrating a biotunable nanoplasmonic optical filter and a highly sensitive few-layer molybdenum disulfide (MoS2) photoconductive component, which can serve as a generic device platform to meet the need of rapid cytokine detection with high sensitivity. The nanoplasmonic filter consists of anticytokine antibody-conjugated gold nanoparticles on a SiO2 thin layer that is placed 170 μm above a few-layer MoS2 photoconductive flake device. The principle of the biosensor operation is based on tuning the delivery of incident light to the few-layer MoS2 photoconductive flake thorough the nanoplasmonic filter by means of biomolecular surface binding-induced LSPR shifts. The tuning is dependent on cytokine concentration on the nanoplasmonic filter and optoelectronically detected by the few-layer MoS2 device. Using the developed optoelectronic biosensor, we have demonstrated label-free detection of IL-1β, a pro-inflammatory cytokine, with a detection limit as low as 250 fg/mL (14 fM), a large dynamic range of 106, and a short assay time of 10 min. The presented biosensing approach could be further developed and generalized for point-of-care diagnosis, wearable bio/chemical sensing, and environmental monitoring.

Cyclewise Operation of Printed MoS2 Transistor Biosensors for Rapid Biomolecule Quantification at Femtomolar Levels

Field-effect transistors made from MoS2 and other emerging layered semiconductors have been demonstrated to be able to serve as ultrasensitive biosensors. However, such nanoelectronic sensors still suffer seriously from a series of challenges associated with the poor compatibility between electronic structures and liquid analytes. These challenges hinder the practical biosensing applications that demand rapid, low-noise, highly specific biomolecule quantification at femtomolar levels. To address such challenges, we study a cyclewise process for operating MoS2 transistor biosensors, in which a series of reagent fluids are delivered to the sensor in a time-sequenced manner and periodically set the sensor into four assay-cycle stages, including incubation, flushing, drying, and electrical measurement. Running multiple cycles of such an assay can acquire a time-dependent sensor response signal quantifying the reaction kinetics of analyte-receptor binding. This cyclewise detection approach can avoid the liquid-solution-induced electrochemical damage, screening, and nonspecific adsorption to the sensor and therefore improves the transistor sensors durability, sensitivity, specificity, and signal-to-noise ratio. These advantages in combination with the inherent high sensitivity of MoS2 biosensors allow for rapid biomolecule quantification at femtomolar levels. We have demonstrated the cyclewise quantification of Interleukin-1β in pure and complex solutions (e.g., serum and saliva) with a detection limit of ∼1 fM and a total detection time ∼23 min. This work leverages the superior properties of layered semiconductors for biosensing applications and advances the techniques toward realizing fast real-time immunoassay for low-abundance biomolecule detection.

Nanofluidic flow assisted assembly of dispersed plasmonic nanostructures into shallow nanochannel sensors

The authors present a method for assembling plasmonic nanostructures into already-sealed shallow nanochannel-based nanofluidic sensor structures. This method is termed as nanofluidic-flow-assisted-assembly (NFAA). NFAA utilizes nanofluidic flows with large shear rate and stress to deposit high-areal-density, well-dispersed plasmonic nanoparticles (NPs) into shallow nanochannel sensing areas. In particular, in a NFAA process, the nano/microfluidic structures are first patterned into a Si or SiO2 substrate and permanently sealed with fused quartz coverslips using plasma sealing. Afterward, a colloidal solution of plasmonic NPs is driven into the shallow nanochannel structures. In the shallow nanochannel areas, the large shear rate and stress of the nanofluidic colloidal solution flow results in the deposition of well-dispersed NPs and effectively prevents undesirable aggregation of NPs. Using NFAA, the authors have demonstrated the deposition of well-dispersed Au NPs with various areal densities (102–104 μm...

Optofluidic cellular immunofunctional analysis by localized surface plasmon resonance

Cytokine secretion assays provide the means to quantify intercellular-signaling proteins secreted by blood immune cells. These assays allow researchers and clinicians to obtain valuable information on the immune status of the donor. Previous studies have demonstrated that localized surface plasmon resonance (LSPR) effects enable label-free, real-time biosensing on a nanostructured metallic surface with simple optics and sensing tunability. However, limited sensitivity coupled with a lack of sample handling capability makes it challenging to implement LSPR biosensing in cellular functional immunoanalysis based on cytokine secretion assay. This paper describes our recent progress towards full development of a label-free LSPR biosensing technique to detect cell-secreted tumor necrosis factor (TNF)-α cytokines in clinical blood samples. We integrate LSPR bionanosensors in an optofluidic platform capable of handling target immune cells in a microfluidic chamber while readily permitting optical access for cytokine detection.