Sub-nanoliter Volume Research Articles

High-sensitivity and fast-response fiber optic temperature sensor using an anti-resonant reflecting optical waveguide mechanism

Temperature sensing is essential for human health monitoring. High-sensitivity (>1 nm/°C) fiber sensors always require long interference paths and temperature-sensitive materials, leading to a long sensor and thus slow response (6–14 s). To date, it is still challenging for a fiber optic temperature sensor to have an ultrafast (∼ms) response simultaneously with high sensitivity. Here, a side-polished single-mode/hollow/single-mode fiber (SP-SHSF) structure is proposed to meet the challenge by using the length-independent sensitivity of an anti-resonant reflecting optical waveguide mechanism. With a polydimethylsiloxane filled sub-nanoliter volume cavity in the SP-SHSF, the SP-SHSF exhibits a high temperature sensitivity of 4.223 nm/°C with a compact length of 1.6 mm, allowing an ultrafast response (16 ms) and fast recovery time (176 ms). The figure of merit (FOM), defined as the absolute ratio of sensitivity to response time, is proposed to assess the comprehensive performance of the sensor. The FOM of the proposed sensor reaches up to 263.94 (nm/°C)/s, which is more than two to three orders of magnitude higher than those of other temperature fiber optic sensors reported previously. Additionally, a three-month cycle test shows that the sensor is highly robust, with excellent reversibility and accuracy, allowing it to be incorporated with a wearable face mask for detecting temperature changes during human breathing. The high FOM and high stability of the proposed sensing fiber structure provide an excellent opportunity to develop both ultrafast and highly sensitive fiber optic sensors for wearable respiratory monitoring and contactless in vitro detection.

One-Step RT-qPCR for Viral RNA Detection Using Digital Analysis.

The rapid detection of viruses is becoming increasingly important to prevent widespread infections. However, virus detection via reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is time-consuming, as it involves independent nucleic acid extraction and complementary DNA synthesis. This process limits the potential for rapid diagnosis and mass analysis, which are necessary to curtail viral spread. In this study, a simple and rapid thermolysis method was developed to circumvent the need for extraction and purification of viral RNA. The developed protocol was applied to one-chip digital PCR (OCdPCR), which allowed thermolysis, RT, and digital PCR in a single unit comprising 20,000 chambers of sub-nanoliter volume. Two viruses such as tobacco mosaic virus and cucumber mosaic virus were tested as model viral particles. First, the temperature, exposure time, and template concentration were optimized against tobacco mosaic viral particles, and the most efficient conditions were identified as 85°C, 5 min, and 0.01 μg/nL with a cycle threshold of approximately 33. Finally, the OCdPCR analysis yielded 1,130.2 copies/µL using 10−2 μg/nL of viral particles in a 30 min thermolysis-RT reaction at 70°C. This novel protocol shows promise as a quick, accurate, and precise method for large-scale viral analysis in the future.

Recent advances in droplet microfluidics for microbiology

Advances in microbiology rely on innovations in technology. Droplet microfluidics, as a versatile and powerful technique that allows high-throughput generation and manipulation of subnanoliter volume droplets, has become an indispensable tool shifting experimental paradigms in microbiology. Droplet microfluidics has opened new avenues to various microbiological research, from resolving single-cell heterogeneity to investigating spatiotemporal dynamics of microbial communities, from precise quantitation of microbiota to systematic decipherment of microbial interactions, and from isolating rare and uncultured microbes to improving genetic engineered strains. In this review, we present recent advances of droplet microfluidics in various fields of microbiology: i) microbial cultivation, ii) microorganism detection and characterization, iii) antibiotic susceptibility testing, iv) microbial interactions, v) microbial biotechnology. We also provide our perspectives on the challenges and future directions for droplet microfluidic-based microbiology research.

Scalable microresonators for room-temperature detection of electron spin resonance from dilute, sub-nanoliter volume solids.

We report a microresonator platform that allows room temperature detection of electron spins in volumes on the order of 100 pl, and demonstrate its utility to study low levels of dopants in perovskite oxides. We exploit the toroidal moment in a planar anapole, using a single unit of an anapole metamaterial architecture to produce a microwave resonance exhibiting a spatially confined magnetic field hotspot and simultaneously high quality-factor (Q-factor). To demonstrate the broad implementability of this design and its scalability to higher frequencies, we deploy the microresonators in a commercial electron paramagnetic resonance (EPR) spectrometer operating at 10 GHz and a NIST-built EPR spectrometer operating at 35 GHz. We report continuous-wave (CW) EPR spectra for various samples, including a dilute Mn2+-doped perovskite oxide, CaTiO3, and a transition metal complex, CuCl22H2O. The anapole microresonator presented here is expected to enable multifrequency EPR characterization of dopants and defects in perovskite oxide microcrystals and other volume-limited materials of technological importance.

Laser-driven self-exfoliation of graphene oxide layers on a fiber facet for Q switching of an Er-doped fiber laser at the longest wavelength

A new method to make an all-fiber nonlinear optic device for laser pulse generation is developed by depositing multi-layer graphene oxide (GO) selectively onto the core of the cleaved fiber facet by combining the electrical arc discharge and the laser-driven self-exfoliation. Using the GO colloid droplet with sub-nanoliter volume, we obtained a GO bulk layer deposited on a fiber facet of the order of milliseconds by using an electric arc. The prepared fiber facet was then included in an Er-doped fiber laser (EDFL) cavity and we obtained a few layers of GO having nonlinear optic two-dimensional (2D) characteristics selectively on the fiber core by the laser-driven self-exfoliation. The 2D GO layers on the fiber core served as a stable and efficient saturable absorber enabling robust pulse train generation at λ = 1600.5 nm , the longest Q -switched laser wavelength in EDFLs. Pulse characteristics were analyzed as we varied the pump power at λ = 980 nm from 105.2 mW to 193.6 mW, to obtain the maximum repetition rate of 17.8 kHz and the maximum output power of 2.3 mW with the minimum pulse duration of 7.8 μs. The proposed method could be further applied to other novel inorganic 2D materials opening a window to explore their novel nonlinear optic laser applications.

Cryo-EM structures from sub-nl volumes using pin-printing and jet vitrification

The increasing demand for cryo-electron microscopy (cryo-EM) reveals drawbacks in current sample preparation protocols, such as sample waste and lack of reproducibility. Here, we present several technical developments that provide efficient sample preparation for cryo-EM studies. Pin printing substantially reduces sample waste by depositing only a sub-nanoliter volume of sample on the carrier surface. Sample evaporation is mitigated by dewpoint control feedback loops. The deposited sample is vitrified by jets of cryogen followed by submersion into a cryogen bath. Because the cryogen jets cool the sample from the center, premounted autogrids can be used and loaded directly into automated cryo-EMs. We integrated these steps into a single device, named VitroJet. The device’s performance was validated by resolving four standard proteins (apoferritin, GroEL, worm hemoglobin, beta-galactosidase) to ~3 Å resolution using a 200-kV electron microscope. The VitroJet offers a promising solution for improved automated sample preparation in cryo-EM studies.

Automated Droplet-Based Microfluidic Platform for Multiplexed Analysis of Biochemical Markers in Small Volumes.

The ability to detect multiple analytes in a small sample volume has significance for numerous areas of research, including organs-on-chip, small animal experiments, and neonatology. The objective of this study was to develop an automated microfluidics platform for multiplexed detection of analytes in microliter sample volumes. This platform employed computer-controlled microvalves to create laminar co-flows of sample and assay reagent solutions. It also contained valve-regulated cross-junction for discretizing sample/reagent mixtures into water-in-oil droplets. Microfluidic automation allowed us to control parameters related to frequency of droplet generation and the number of droplets of the same composition, as well as the size of droplets. Each droplet represented an individual enzymatic assay carried out in a sub-nanoliter (0.8 nL) volume reactor. An enzymatic reaction involving target analyte and assay reagents produced colorimetric or fluorescent signals in droplets. Importantly, intensity of optical signal was proportional to the concentration of analyte in question. This microfluidic bioanalysis platform was used in conjunction with commercial "mix-detect" assays for glucose, total bile acids, and lactate dehydrogenase (LDH). After characterizing these assays individually, we demonstrated sensitive multiplexed detection of three analytes from as little as 3 μL. In fact, this volume was sufficient to generate multiple repeat droplets for each of the three biochemical assays as well as positive control droplets, confirming the quality of assay reagents and negative control droplets to help with background subtraction. One potential application for this microfluidic bioanalysis platform involves sampling cell-conditioned media in organ-on-chip devices. To highlight this application, hepatocyte spheroids were established in microfluidic devices, injured on-chip by exposure to lipotoxic agent (palmitate), and then connected to the bioanalysis module for daily monitoring of changes in cytotoxicity (LDH), energy metabolism (glucose), and liver function (total bile acids). Microfluidic in-droplet assays revealed increased levels of LDH as well as reduction in bile acid synthesis-results that were consistent with hepatic injury. Importantly, these experiments highlighted the fact that in-droplet assays were sufficiently sensitive to detect changes in functional output of a relatively small (∼100) number of hepatocyte spheroids cultured in a microfluidic device. Moving forward, we foresee increasing the multiplexing capability of this technology and applying this platform to other biological/medical scenarios where detection of multiple analytes from a small sample volume is desired.

Sub-Nanoliter Volume Measurement by Using a Digital Holographic Microscope

Sub-Nanoliter Volume Measurement by Using a Digital Holographic Microscope

Detection of phase transition in polyethylene glycol using a multimodal micromechanical acoustic resonator

A piezoelectrically transduced micromechanical beam acoustic resonator is utilized to detect solid to liquid phase transitions in sub-nanoliter volume of Polyethylene Glycol (PEG) 1000. A lower frequency flexural mode and a higher frequency length-extensional mode are simultaneously monitored for changes in the device response. The phase transition of the PEG drop perturbs the acoustic loading of the resonator, bringing about characteristic and repeatable changes in the frequency response. The phase transitions in the drop are verified optically, and the resonant parameters are compared with the control case when the resonator is pristine without the presence of any PEG drop.

Electronic desalting for controlling the ionic environment in droplet-based biosensing platforms.

The ability to control the ionic environment in saline waters and aqueous electrolytes is useful for desalination as well as electronic biosensing. We demonstrate a method of electronic desalting at micro-scale through on-chip micro electrodes. We show that, while desalting is limited in bulk solutions with unlimited availability of salts, significant desalting of ≥1 mM solutions can be achieved in sub-nanoliter volume droplets with diameters of ∼250 μm. Within these droplets, by using platinum-black microelectrodes and electrochemical surface treatments, we can enhance the electrode surface area to achieve >99% and 41% salt removal in 1 mM and 10 mM salt concentrations, respectively. Through self-consistent simulations and experimental measurements, we demonstrate that conventional double-layer theory over-predicts the desalting capacity and, hence, cannot be used to model systems that are mass limited or undergoing significant salt removal from the bulk. Our results will provide a better understanding of capacitive desalination, as well as a method for salt manipulation in high-throughput droplet-based microfluidic sensing platforms.

Exploiting highly ordered subnanoliter volume microcapillaries as microtools for the analysis of antibody producing cells.

The interrogation of highly diverse repertoires of heterogeneous cell populations on a single cell basis increases the likelihood that a cell with unique characteristics will be identified. We have developed a new single cell analysis system comprising millions of bundled subnanoliter volume bioincubation chambers for the identification and recovery of target specific antibody secreting cells (ASCs). This platform integrates dual surface screening with dedicated user driven data analysis and automated cell recovery enabling multiple biophysical parameters to be tracked for millions of antibody leads in parallel. This direct clone analysis and selection technology is a clear deviation from current microfabricated well-based approaches and offers drastically enhanced screening throughput, simultaneous dual surface analysis, and rapid automated single cell recovery. The technology is also applicable to screening both bacterial and mammalian antibody secreting cells. We demonstrate the implementation and feasibility of this platform in identifying target specific antibodies from bacterial, hybridoma, and B cell libraries.

Multi-chamber actuated micro-dispensing with a single nozzle for sub-nanoliter droplet formation

A novel concept of single-nozzle micro-dispensing device with multiple pressurizing chambers is proposed for high-throughput drug screening applications such as arraying new drug candidates with sub-nanoliter volume. The theoretical study with a simplified electrical circuit model of the fluidic system shows that the proposed model is effective to sustain jetting stability at high frequency due to an increase in the natural frequency of the fluidic system and high attenuation of the negative pressure wave in the fluidic system. The fabricated device was able to form uniform droplets at up to 7 kHz having 115 pL (1.15 × 10−10 L) in volume and 1.8 m s−1 ∼ 2.5 m s−1 in velocity.

Method for physiologic phenotype characterization at the single-cell level in non-interacting and interacting cells

Intercellular heterogeneity is a key factor in a variety of core cellular processes including proliferation, stimulus response, carcinogenesis, and drug resistance. However, cell-to-cell variability studies at the single-cell level have been hampered by the lack of enabling experimental techniques. We present a measurement platform that features the capability to quantify oxygen consumption rates of individual, non-interacting and interacting cells under normoxic and hypoxic conditions. It is based on real-time concentration measurements of metabolites of interest by means of extracellular optical sensors in cell-isolating microwells of subnanoliter volume. We present the results of a series of measurements of oxygen consumption rates (OCRs) of individual non-interacting and interacting human epithelial cells. We measured the effects of cell-to-cell interactions by using the system's capability to isolate two and three cells in a single well. The major advantages of the approach are: 1. ratiometric, intensity-based characterization of the metabolic phenotype at the single-cell level, 2. minimal invasiveness due to the distant positioning of sensors, and 3. ability to study the effects of cell-cell interactions on cellular respiration rates.

Membrane-assisted online renaturation for automated microfluidic lectin blotting.

Aberrant glycosylation plays a pivotal role in a diverse set of diseases, including cancer. A microfluidic lectin blotting platform is introduced to enable and expedite the identification of protein glycosylation based on protein size and affinity for specific lectins. The integrated multistage assay eliminates manual intervention steps required for slab-gel lectin blotting, increases total assay throughput, limits reagent and sample consumption, and is completed using one instrument. The assay comprises non-reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by online post-sizing SDS filtration and lectin-based affinity blotting. Important functionality is conferred through both device and assay advances that enable integration of nanoporous membranes flanking a central microchamber to create sub-nanoliter volume compartments that trap SDS-protein complexes and allow electrophoretic SDS removal with buffer exchange. Recapitulation of protein binding for lectin was optimized through quantitative assessment of SDS-treated green fluorescent protein. Immunoglobulin A1 aberrantly glycosylated with galactose-deficient O-glycans was probed in ~6 min using ~3 μL of sample. This new microfluidic lectin blotting platform provides a rapid and automated assay for the assessment of aberrant glycosylation.

Confined PFSA–zeolite composite membrane for self-humidifying fuel cell

Perfluorosulfonic acid (PFSA) polyelectrolyte confined in subnanoliter volume within zeolite cladded walls exhibits higher glass transition temperature and excellent tolerance to high-temperature fuel cell operation under dry conditions, generating an order of magnitude higher power density than standard PEMFC.

Integration of ground aerogel particles as chromatographic stationary phase into microchip

C16 modified and ground monolithic silica aerogel particles in submicrometer size, as a new type of stationary phase was prepared and integrated in polydimethylsiloxane (PDMS) microchip. The aerogel particles were packed into the microfluidic channel using a simple procedure, which does not require any special frit or fabrication step to retain the particles. The subnanoliter volume of samples can be transported through the porous, short length of packing with low pressure (<3bar). Food dyes as test components could be separated using low pressure within 6s. A 50-fold preconcentration could be achieved by retaining 100nL volume of sample on the packing and elution with methanol.

Sub-nanoliter nuclear magnetic resonance coils fabricated with multilayer soft lithography

We describe the fabrication and characterization of sub-nanoliter volume nuclear magnetic resonance (NMR) transceiver coils that are easily amenable to integration within PDMS-based microfluidics. NMR coils were constructed by the injection of liquid metal into solenoidal cavities created around a microchannel using consecutive replica molding and bonding of PDMS layers. This construction technique permits the integration of NMR coils with solenoidal, toroidal or other three-dimensional geometries within highly integrated microfluidic systems and are one step toward NMR-based chemical screening and analysis on chip. The current proof-of-principle implementation displays limited sensitivity and resolution due to the conductivity and magnetic susceptibilities of the construction materials. However, NMR measurements and finite-element simulations made with the current device geometry indicate that optimization of these materials will allow for the collection of spectra from sub-millimolar concentration samples in less than 1 nL of solution.

An opto-fluidic ring resonator biosensor for the detection of organophosphorus pesticides

Abstract We developed a novel label-free opto-fluidic ring resonator (OFRR) biosensor for detection of an organophosphorus (OP) pesticide. The OFRR is based on a micro-sized glass capillary whose circular wall forms a ring resonator that supports the whispering gallery modes (WGMs). The WGMs has an evanescent field in the capillary core and interacts with the analyte flowing in the capillary. We used parathion-methyl as a model system to investigate the OFRR sensing performance in terms of bulk refractive index sensitivity, surface activation for affinity property, detection limit, and reproducibility. The performance of the OFRR was further compared with that of the Biacore 3000 SPR system. Our results show that the detection limit of 3.8 × 10−11 M for parathion-methyl was achieved with an analysis time of about 0.5 min, 10 times faster than the surface plasmon resonance (SPR) system. Furthermore, the OFRR biosensor demonstrated excellent reproducibility (R.S.D. = 3.5%, n = 5). The OFRR offers optical label-free detection mechanism with integrated microfluidics. It is a promising technology platform for development of portable multi-channel biosensors with high sensitivity, quick detection time, and sub-nanoliter detection volume.

Label-free quantitative DNA detection using the liquid core optical ring resonator

We demonstrated quantitative real-time label-free detection of DNA sequences using the liquid core optical ring resonator (LCORR) sensor. The LCORR is a recently developed sensing platform that integrates microfluidics and photonic sensing technology with low detection limit and sub-nanoliter detection volume. We analyzed experimentally and theoretically the LCORR response to a variety of DNA samples that had different strand lengths (25–100 bases), number of base- mismatches (1–5), and concentrations (10 pM to 10 μM) to evaluate the LCORR sequence detection capability. In particular, we established the linear correlation between the LCORR sensing signal and the molecule density, which allows us to accurately calculate the molecule density on the surface. It is found that the probe surface coverage was 26–51% and the extent of hybridization was 40–50%. The titration curve for 25-base probe and 25-base target DNA yields a dissociation constant of 2.9 nM. With a 37.1 nm/RIU LCORR, detection of 10 pM bulk DNA concentration was demonstrated. The mass detection limit was estimated to be 4 pg/mm 2, corresponding to a density of 10 10 molecules/cm 2 on the surface. We also showed that the LCORR was sensitive enough to differentiate DNA with only a few base-mismatches based on the raw sensing signal and kinetic analysis. Our work will provide important insight into the light-DNA interaction at the ring resonator surface and lay a foundation for future LCORR-based DNA label-free microarray development.

A microfluidic device to confine a single cardiac myocyte in a sub-nanoliter volume on planar microelectrodes for extracellular potential recordings.

A hybrid chip is described which combines a microfluidic network fabricated in a silicone elastomer (PDMS) with planar microelectrodes. It was used to measure extracellular potentials from single adult murine cardiac myocytes in a restricted extracellular space. The recorded variations in the extracellular potentials were caused by transmembrane currents associated with spontaneously initiated intracellular calcium waves. Single cells were trapped inside the 100 pl microchamber by pressure gradients and maintained for several hours by continuous perfusion. In addition, the localized delivery of drugs to a portion of the cell was demonstrated. The impedance of the electrodes was reduced by a factor of 10 to 20 after the electrodeposition of platinum black. Biopotentials recorded from single cells with platinum black electrodes showed a three-fold decrease in the noise, resulting in a maximum signal-to-noise ratio of 15:1. Characteristic variations in the frequency and shape of the extracellular potentials were observed among different cells which are most likely due to differences in the sarcoplasmic reticulum Ca(2+) load. Our device architecture permits the integration of electrochemical and optical sensors for multiparameter recordings.