Ultrasmall Sample Volumes Research Articles

Label-free and specific detection of active Botulinum neurotoxin in 0.5 μL drops with the meta-nano-channel field-effect biosensor

Botulinum neurotoxins (BoNT) are the most poisonous and lethal substances known to humans, and as such there is a great need for fast, easy-to-use, and early BoNT detection technologies which are not available today. A biosensor based on field-effect transistor (FET) is a highly pursued sensing platform due to the inherent low-noise and amplification, low-cost, and the ultimate potential for the sensing of multiple analytes from an ultra-small sample volume. In the current study we employ the novel Meta-Nano-Channel (MNC) FET biosensor for specific and label-free sensing of BoNT. The MNC biosensor is a multi-channel device which provides the means to optimize the coupling between the biological interactions and the readout sensing current, by applying a tight control over both the electrodynamics of the sensing current and the electrostatics of the biological interactions. Importantly, the MNC FET biosensor presents excellent stability, repeatability and robustness in wet conditions. Finally, the multi-channel approach of the MNC biosensor allows for tunable sensing performance in terms of limit-of-detection, sensitivity and dynamic range.

Diagnosis of Parkinson's Disease via the Metabolic Fingerprint in Saliva by Deep Learning.

Parkinson's disease (PD) is the second cause of the neurodegenerative disorder, affecting over 6 million people worldwide. The World Health Organization estimated that population aging will cause global PD prevalence to double in the coming 30 years. Optimal management of PD shall start at diagnosis and requires both a timely and accurate method. Conventional PD diagnosis needs observations and clinical signs assessment, which are time-consuming and low-throughput. A lack of body fluid diagnostic biomarkers for PD has been a significant challenge, although substantial progress has been made in genetic and imaging marker development. Herein, a platform that noninvasively collects saliva metabolic fingerprinting (SMF) by nanoparticle-enhanced laser desorption-ionization mass spectrometry with high-reproducibility and high-throughput, using ultra-small sample volume (down to 10 nL), is developed. Further, excellent diagnostic performance is achieved with an area-under-the-curve of 0.8496 (95% CI: 0.7393-0.8625) by constructing deep learning model from 312 participants. In conclusion, an alternative solution is provided for the molecular diagnostics of PD with SMF and metabolic biomarker screening for therapeutic intervention.

Single‐Cell Metabolomics by Mass Spectrometry: Opportunities and Challenges

AbstractCellular heterogeneity is an inherent property of cell populations with a wide spectrum of biological manifestations, ranging from barely observable variations that enhance organismal adaptation, to life‐threatening differences. Single‐cell metabolomics can reveal molecular information and variations in metabolite concentrations between cells that are masked in cell‐population studies. These differences are quantitatively captured by the abundance distributions for the population and their statistical analysis can reveal the presence of latent subpopulations. In recent years, mass spectrometry (MS), combined with novel sampling and ionization techniques, has become an important tool for single‐cell metabolomics. These new techniques must contend with significant challenges in the form of small cell sizes and volumes, ultratrace metabolite amounts per cell, and potentially interfering high turnover rates and rapid diffusion. Owing to the ultrasmall sample volume, low abundance of some metabolites, and poor ionization efficiencies, metabolite detection, identification, and accurate quantitation remain a major challenge. The ability of some techniques to analyze tissue‐embedded cells opens the door for spatial metabolomics, potentially revealing cellular synergism in organ level metabolism. In this Concept article, we present a bird's eye view of these major themes in single cell metabolomics and some of the relevant MS‐based methods.

Tracking Brownian motion in three dimensions and characterization of individual nanoparticles using a fiber-based high-finesse microcavity

The dynamics of nanosystems in solution contain a wealth of information with relevance for diverse fields ranging from materials science to biology and biomedical applications. When nanosystems are marked with fluorophores or strong scatterers, it is possible to track their position and reveal internal motion with high spatial and temporal resolution. However, markers can be toxic, expensive, or change the object’s intrinsic properties. Here, we simultaneously measure dispersive frequency shifts of three transverse modes of a high-finesse microcavity to obtain the three-dimensional path of unlabeled SiO2 nanospheres with 300 μs temporal and down to 8 nm spatial resolution. This allows us to quantitatively determine properties such as the polarizability, hydrodynamic radius, and effective refractive index. The fiber-based cavity is integrated in a direct-laser-written microfluidic device that enables the precise control of the fluid with ultra-small sample volumes. Our approach enables quantitative nanomaterial characterization and the analysis of biomolecular motion at high bandwidth.

Nanochannel chromatography and photothermal optical diffraction: Femtoliter sample separation and label-free zeptomole detection

Column miniaturization of liquid chromatography is a major trend in separation sciences with the advent of single-cell proteomics and metabolomics. Nanochannel chromatography is one of the promising tools for single-cell analyses because it provides ultra-small sample volume and high separation efficiency. However, non-fluorescent molecular detection in such small channels is still quite difficult due to fL–aL sample volume, which hinders further miniaturization of nanochannels. In this study, we overcame the size limitation of nanochannel chromatography by our label-free molecular detection method: photothermal optical diffraction (POD), which utilizes the photothermal effect of analytes and optical diffraction by nanochannel. The combination of the nanochannel chromatography and the POD enables 1.8 fL sample separation and label-free molecule detection in nanochannels with 800 nm width and 300 nm depth at the optimized experimental conditions. The limit of detection is 5.4 zmol (3300 molecules), approximately 50 times lower than the conventional label-free detection method. Furthermore, the theoretical plate number is calculated to be 105 plates/m, and the separation performance is discussed. Our label-free detection method will be widely used as a universal detector for nanochannel chromatography.

Controllable fabrication of pico/femtoliter pipette sampling probes and visual sample volume determination

We propose a hydraulically assisted eddy-current etching method for the controllable fabrication of pico/femtoliter sampling probes with equal inner diameters along the length of the probe. The relative standard deviations of the outer and inner diameters (O.D. and I.D., respectively) of several 1.07-μm-I.D. sampling probe tips fabricated in a single batch using this method were 2.2% and 2.8%, respectively, and the average O.D./I.D. ratio of these probes was 1.17. The probe fabrication method has high reproducibility, and sharp tips are produced, which is advantageous for the transfer of ultra-small sample volumes. Further, the narrow, equal diameter, cylindrical inner bore allows the online, visual determination of the pipetted sample volume by utilizing a microscopic imaging system to measure the liquid length. This value is converted linearly to the pipetted volume, whose measurement error is on a sub-pixel level. Image-based sampling using the fabricated probes was achieved by connecting the probe to an electroosmotic pump, which allowed the controlled pipetting of pico/femtoliter samples. Because the pipette allows samples of the same volume to be measured accurately, the pipette was applied for the semiquantitative mass spectrometry analysis of the metabolites in individual epidermal cells sampled from different parts of Portulaca oleracea plants. The results show that different types of cells have distinct metabolite profiles. Further, the experiments showed that, in dopamine-rich leaves, the content of dopamine in the petioles is higher than that in the foliage. The probe fabrication strategy opens new avenues for the controllable pipetting of ultra-small volume samples and the realization of the visual pipetting of pico/femtoliter samples.

Sensitive and ultrasmall sample volume gas sensor based on a sealed slot waveguide.

Taking advantage of the near-infrared (IR) absorption characteristics of gases, a sensor with an ultrasmall sample volume composed of a sealed slot waveguide and based on evanescent field absorption is proposed in this paper. Compared with a traditional open-slot waveguide, it features small volume antiparticles depositing pollution over the long-term and is insensitive to surroundings. Working at 1645nm, a large evanescent field ratio of 0.27 is obtained by simulation and optimization; meanwhile, the propagation loss is around 1.6dB/cm. The needed sample volume of the designed sensor under the structure parameters of w_air=40 nm, h_air=400 nm, and waveguide length=3 cm is approximately 480 μm3, which helps the sensor demonstrate excellent performance for gas analysis with an ultrasmall sample volume.

Microfluidic Techniques for Analytes Concentration

Microfluidics has been undergoing fast development in the past two decades due to its promising applications in biotechnology, medicine, and chemistry. Towards these applications, enhancing concentration sensitivity and detection resolution are indispensable to meet the detection limits because of the dilute sample concentrations, ultra-small sample volumes and short detection lengths in microfluidic devices. A variety of microfluidic techniques for concentrating analytes have been developed. This article presents an overview of analyte concentration techniques in microfluidics. We focus on discussing the physical mechanism of each concentration technique with its representative advancements and applications. Finally, the article is concluded by highlighting and discussing advantages and disadvantages of the reviewed techniques.

Optical integrated chips with micro and nanostructures for refractive index and SERS-based optical label-free sensing

Abstract: Label-free optical biosensing technologies have superior abilities of quantitative analysis, unmodified targets, and ultrasmall sample volume, compared to conventional fluorescence-label-based sensing techniques, in detecting various biomolecules. In this review article, we introduce our recent results in the field of evanescent-wavebased refractive index sensing and surface enhanced Raman scattering (SERS)-based sensing, both of which are promising platforms for label-free optical biosensors. First, silicon-on-insulator (SOI) nanowire waveguide and metallic surface plasmon resonance (SPR)-based refractive index sensing are discussed. In order to improve the detection limit, phase interrogation techniques are introduced to these types of sensors based on prism-coupled SPR and SOI microring resonators. A detection limit in the order of 10−6 refractive index unit is achieved. Detection of 16.7 pM anti-IgG is also demonstrated based on the SPR devices. Second, SERS substrates based on various nanometallic structures are discussed. Metallic nanowire arrays and inverted nanopyramids and grooves with a thin metal surface are fabricated based on anisotropic wetetching of silicon substrates. Both structures have demonstrated a Raman signal enhancement on the order of 107. In order to improve the extraction efficiency of the Raman signal at a high wave number, a nano-bowtie array substrate is fabricated, which exhibits double resonances at both the excitation wavelength and the desired Raman scattering wavelength. Experimental results have shown that this double-resonance structure can further enhance the received Raman signal, as compared to conventional SERS substrates with only one resonance at the excitation wavelength.

Determination of Effective Stability Constants of Ion-Carrier Complexes in Ion Selective Nanospheres with Charged Solvatochromic Dyes.

Ionophores are widely used ion carriers in ion selective sensors. The effective stability constant (β) is a key physical parameter providing valuable guidelines to the design of ionophores and carrier-based ion selective sensors. The β value of ion-carrier complex in plasticized poly(vinyl chloride) (PVC) membranes and solutions have been determined in the past by various techniques, but most of them are difficult to implement at the nanoscale owing to the ultrasmall sample volume. A new methodology based on charged solvatochromic dyes is introduced here for the first time to determine β values directly within ion selective nanospheres. Four ionophores with different selectivities toward Na(+), K(+), Ca(2+), and H(+), respectively, are successfully characterized in nanospheres composed of triblock copolymer Pluronic F-127 and bis(2-ethylhexyl) sebacate. The values determined in the nanospheres are smaller compared with those in plasticized PVC membranes, indicating a more polar nanosphere microenvironment and possible uneven distribution of the sensing components in the interfacial region.

Temperature-Controlled Electrochemical Microsensors and Microsensor Arrays

There is a growing demand for rapid, simple, and low-cost chemical sensors for next generation bio-manufacturing, medical diagnostics and environmental monitoring. Current reliable technologies like mass spectrometry and fluorescence-based assays suffer from low throughput and relatively high cost. A preferred alternative method would use electrochemical techniques, which can be adapted for automated screening of multiple chemical/biochemical targets. Unfortunately, due to the complexity of real-world samples, which can contain multiple interfering species spanning several orders of magnitude in concentration, the broad range of electrochemical sensing strategies found in the literature have produced only limited commercial technologies. Actively controlling an electrode’s surface temperature, which is simultaneously one of the most critical and most neglected parameters in determining sensor response, offers a route towards improved electrochemical detection for real-world samples. Previous work has demonstrated certain advantages of both directly and indirectly heated electrodes in terms of sensitivity and selectivity[1,2]. In this work we present the design concepts, fabrication methods, and temperature characterization approaches for microfabricated, temperature-controlled electrodes. The use of modern microfabrication techniques provides several advantages by facilitating the construction of sensors with highly reproducible heating/electrochemical characteristics, the production of high spatial-density sensor arrays, and the compatibility of sensors with complementary microfluidic/MEMS components for sensing in ultra-small sample volumes. The sequential deposition of patterned metallic structures and insulating layers (e.g. SiO2) was used to fabricate Au disk electrodes (r ≤ 75 μm) with underlying platinum resistive heating elements as shown in Figure 1A. The small vertical electrode-heater separation (≲ 500 nm) allows for rapid and efficient heating of the electrode surface. Infrared and fluorescent temperature imaging techniques are used to evaluate temperature gradients and thermal response times, which impact sensor performance. We demonstrate temperature-dependent electrochemical measurements performed on model systems (e.g. Fe(CN)6 3-/4- and Ru(NH3)6 3+/2+) in which coupled heat and mass transport impart signal enhancement as shown in the cyclic voltammograms in Figure 1B. We also discuss the impact of various temperature control routines, such as temperature steps, pulses and oscillations, on electrochemical signal content. References (1) Gründler, P. Fresenius. J. Anal. Chem. 2000, 367, 324–328. (2) Flechsig, G.-U.; Walter, A. Electroanalysis 2012, 24, 23–31. Figure 1

Spatiotemporal lipid profiling during early embryo development of Xenopus laevis using dynamic ToF-SIMS imaging

Time-of-flight secondary ion mass spectrometry (ToF-SIMS) imaging has been used for the direct analysis of single intact Xenopus laevis embryo surfaces, locating multiple lipids during fertilization and the early embryo development stages with subcellular lateral resolution (∼4 μm). The method avoids the complicated sample preparation for lipid analysis of the embryos, which requires selective chemical extraction of a pool of samples and chromatographic separation, while preserving the spatial distribution of biological species. The results show ToF-SIMS is capable of profiling multiple components (e.g., glycerophosphocholine, SM, cholesterol, vitamin E, diacylglycerol, and triacylglycerol) in a single X. laevis embryo. We observe lipid remodeling during fertilization and early embryo development via time course sampling. The study also reveals the lipid distribution on the gamete fusion site. The methodology used in the study opens the possibility of studying developmental biology using high resolution imaging MS and of understanding the functional role of the biological molecules.

Trace-Gas Detection with Off-Beam Quartz Enhanced Photoacoustic Spectroscopy

Trace-gas sensors have a wide range of potential applications such as environmental monitoring, climate research, agriculture, workplace safety, medical diagnostics, and industrial process control. A recently introduced technique called quartz-enhanced photoacoustic spectroscopy (QEPAS) is described. QEPAS use a quartz tuning fork as an acoustic transducer for a photoacoustic signal induced in an absorbing gas by modulated optical radiation. Advantages of the QEPAS compared to conventional photoacoustic spectroscopy include immunity to environmental acoustic noise and ultra-small sample volume. Trace gases of \(\hbox {CH}_{4}, \hbox {H}_{2}\hbox {O}\), and \(\hbox {NO}_{2}\) were monitored with a novel off-beam QEPAS approach and are described in detail.

Microanalytical study of sub-nanoliter samples by capillary electrophoresis – mass spectrometry with 100 % injection efficiency

A method is presented for conducting fast capillary electrophoresis (CE) with ultrasmall sample volumes (<10 nL) and subsequent determination of target analytes by time-of-flight mass spectrometry (TOF-MS). Furthermore, an approach towards preconcentration by solvent evaporation of samples as small as 500 nL is shown. Ultrasmall samples of biologically important cyclic nucleotides (cyclic guanosine monophosphate, cyclic adenosine monophosphate, cyclic cytidine monophosphate) served as model compounds. Sample volumes as small as 500 pL were used to perform sample uptake and CE injections by capillary batch injection (CBI). This resulted in detection limits between 2 and 5 fmol. The fast rate of sample evaporation prevents the operation of nano-/subnano-liter samples under room temperature conditions. A CBI-CE-TOF-MS setup incorporating a Peltier element and a thermostat was used for the handling of very small samples. In combination with tapered fused silica injection capillaries and the high-precision guiding system of the CBI setup, nearly 100 % efficient injections of ultrasmall samples into funnelled separation capillaries could be managed. The overall system allowed for high-throughput CE separations from densely arranged sample droplets or vials with RSD of peak heights ranging from 6 to 8 % (n = 5). CE-TOF-MS separations of mixtures of cyclic nucleotides were carried out in capillaries as short as 20 cm (25 μm ID) in less than 55 s with high separation efficiency. We report details of the setup and optimizations with respect to etching of capillaries (tapering and funnelling), sample uptake and injection as well as separation of the model compounds. Moreover, the capabilities of the experimental setup for preconcentration of target analytes by solvent evaporation are described. Preconcentration factors of up to 100 could be achieved. This approach was successfully used for the determination of the cyclic nucleotide cGMP in (spiked) human urine samples.

Microbeam, timing and signal-resolved studies of nuclear materials with synchrotron X-ray sources

The development of ultra-brilliant synchrotron X-ray sources enables characterization methods that are particularly important for nuclear materials. Here we discuss emerging synchrotron methods with unprecedented signal-to-noise, spatial and time resolution. Microprobe methods are discussed that extend virtually any X-ray characterization measurement to ultra-small sample volumes. This ability is critical to resolve heterogeneities in nuclear materials and for studies on volumes with vastly lower activity than are needed for traditional X-ray characterization. Specific methods discussed include microdiffraction for the characterization of local crystal structure and micro-spectroscopy techniques that allow for characterization of elemental distributions with sensitivity for daughter products, oxidation states and diffusion through buffer layers. Opportunities are also discussed that exploit the high brilliance and pulsed nature of synchrotron radiation to reduce backgrounds from sample radiation and to study materials dynamics.

Metabolic Profiling of Ultrasmall Sample Volumes with GC/MS: From Microliter to Nanoliter Samples

Profiling of metabolites is increasingly used to study the functioning of biological systems. For some studies the volume of available samples is limited to only a few microliters or even less, for fluids such as cerebrospinal fluid (CSF) of small animals like mice or the analysis of individual oocytes. Here we present an analytical method using in-liner silylation coupled to gas chromatography/mass spectrometry (GC/MS), that is suitable for metabolic profiling in ultrasmall sample volumes of 2 microL down to 10 nL. Method performance was assessed in various biosamples. Derivatization efficiencies for sugars, organic acids, and amino acids were satisfactory (105-120%), and repeatabilities were generally better than 15%, except for amino acids that had repeatabilities up to about 35-40%. For endogenous sugars and organic acids in fetal bovine serum, the response was linear for aliquots from 10 nL up to at least 1 microL. The developed GC/MS method was applied for the analysis of different sample matrixes, i.e., fetal bovine serum, mouse CSF, and aliquots of the intracellular content of Xenopus laevis oocytes. To the best of our knowledge, we present here the first comprehensive GC/MS metabolite profiles from mouse CSF and from the intracellular content of a single X. laevis oocyte.

Analysis of hollow-core photonic bandgap fibers for evanescent wave biosensing

Hollow-core photonic bandgap fiber (HC-PBGF)-based evanescent wave biosensors are demonstrated and analyzed theoretically and experimentally. With 95% of the guided light power residing in the samples, the measured absorbance for a 30-cm-long fiber filled with a 0.2 microM Alexa Fluor 700-labeled DNA Oligo solution is 1.06. This is in good agreement with the theoretical prediction, which is evaluated by using the refractive index scaling law. The HC-PBGFs thus offer both efficiency and simplicity for the detection of biomolecules in ultra-small sample volumes.

Identification of Herb Acanthopanax senticosus (Rupr. Et Maxim.) Harms by Capillary Electrophoresis with Electrochemical Detection

In this paper, a rapid, high efficient, sensitive and inexpensive approach based on a combination of simple ultrasonic extract and capillary electrophoresis (CE) separation with electrochemical detection (ED), is described to identify herbs by comparing their CE-ED profiles (namely, CE-ED electropherograms). The proposed method takes advantage of ultra-small sample volume, low consumption of organic solvent, simple sample pretreatment and easy cleanup procedure. It was applied to analyze the CE-ED profiles of stems of herb Acanthopanax senticosus (Rupr. Et Maxim.) Harms from different sources and different parts (roots, rhizomes, stems and leaves) of this herb. By comparing peak number, peak height and peak height ratio, we found that the CE-ED profiles showed big differences for the herbs from the different sources and the different parts of this herb. In addition, the distribution of bioactive compounds (isofraxidin, rutin and chlorogenic acid) in the different parts of this herb and their content variations affected by the source were studied with the CE-ED method. Based on their own unique CE-ED profiles, these herbs from the different sources and the different parts of this herb could be easily distinguished. Therefore, the proposed approach could be used as a rapid, high efficient and sensitive method for the identification of herbal medicines.

SPCE Based Glucose Sensor Employing Novel Thermostable Glucose Dehydrogenase, FADGDH: Blood Glucose Measurement with 150nL Sample in One Second

Self-monitoring of blood glucose (SMBG) is an important component of the modern therapy for diabetes mellitus. Thanks to the current progress in electronics and sensor fabrication technology, both the time and the blood sample volume required for the measurement have decreased drastically. However, devices that work with an even smaller sample volume and a shorter measurement time are in demand. A disposable glucose sensor that works with an ultra-small sample volume was developed employing the novel thermostable glucose-dehydrogenase (FADGDH) complex composed of a catalytic subunit, an electron transfer subunit (cytochrome c), and a small subunit. The electrode is a screen-printed carbon electrode (SPCE), and hexaammineruthenium (III) chloride (Ru complex) is utilized as the electron mediator. A disposable enzyme sensor was constructed by depositing the FADGDH complex and Ru complex onto the SPCE, and the sensor performance was evaluated. Whole-blood glucose can be measured within 1 sec using this enzyme sensor and a 150-nL whole-blood sample, with high precision (>0.99br>) and high reproducibility (CV<0.45%br>) within the glucose concentration range of 0-533 mg/dL. The sensor reading was stable for more than 60 days even at 70 degrees C. The simplicity of the construction and the high precision of this FADGDH-based glucose biosensor makes it an alternative to previously reported commercially available glucose sensors. Especially the sample volume of 150 nL and the 1-sec measurement time are the highest specifications in the world for currently available glucose sensors designed for the SMBG.

Selective detection of antibodies in microstructured polymer optical fibers

We demonstrate selective detection of fluorophore labeled antibodies from minute samples probed by a sensor layer of complementary biomolecules immobilized inside the air holes of microstructured Polymer Optical Fiber (mPOF). The fiber core is defined by a ring of 6 air holes and a simple procedure was applied to selectively capture either alpha-streptavidin or alpha-CRP antibodies inside these air holes. A sensitive and easy-to-use fluorescence method was used for the optical detection. Our results show that mPOF based biosensors can provide reliable and selective antibody detection in ultra small sample volumes.