Ultra-small Sample Research Articles

Rapid micromotor-based naked-eye immunoassay

A dynamic micromotor-based immunoassay, exemplified by cortisol detection, based on the use of tubular micromotors functionalized with a specific antibody is described. The use of antibody-functionalized micromotors offers huge acceleration of both direct and competitive cortisol immunoassays, along with greatly enhanced sensitivity of direct and competitive immunoassays. The dramatically improved speed and sensitivity reflect the greatly increased likelihood of antibody-cortisol contacts and fluid mixing associated with the dynamic movement of these microtube motors and corresponding bubble generation that lead to a highly efficient and rapid recognition process. Rapid naked-eye detection of cortisol in the sample is achieved in connection to use of horseradish peroxidase (HRP) tag and TMB/H2O2 system. Key parameters of the competitive immunoassay (e.g., incubation time and reaction volume) were optimized. This fast visual micromotor-based sensing approach enables “on the move” specific detection of the target cortisol down to 0.1μgmL−1 in just 2min, using ultrasmall (50µL) sample volumes.

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.

Phase Concentration Determination of Fe16N2 Using State of the Art Neutron Scattering Techniques

Due to limitations on the availability of rare earth elements it is imperative that new high energy product rare earth free permanent magnet materials are developed for the next generation of energy systems. One promising low cost permanent magnet candidate for a high energy magnet is α″-Fe16N2, whose giant magnetic moment has been predicted to be well above any other from conventional first principles calculations. Despite its great promise, the α″ phase is metastable; making synthesis of the pure phase difficult, resulting in less than ideal magnetic characteristics. This instability gives way to a slew of possible secondary phases (i.e. α-Fe, Fe2O3, Fe8N, Fe4N, etc.) whose concentrations are difficult to detect by conventional x-ray diffraction. Here we show how high resolution neutron diffraction and polarized neutron reflectometry can be used to extract the phase concentration ratio of the giant magnetic phase from ultra-small powder sample sizes (~0.1 g) and thin films. These studies have led to the discovery of promising fabrication methods for both homogeneous thin films, and nanopowders containing the highest reported to date (>95%) phase concentrations of room temperature stable α″-Fe16N2.

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.

A superhydrophobic chip based on SU-8 photoresist pillars suspended on a silicon nitride membrane.

We developed a new generation of superhydrophobic chips optimized for probing ultrasmall sample quantities by X-ray scattering and fluorescence techniques. The chips are based on thin Si3N4 membranes with a tailored pattern of SU-8 photoresist pillars. Indeed, aqueous solution droplets can be evaporated and concentrated at predefined positions using a non-periodic pillar pattern. We demonstrated quantitatively the deposition and aggregation of gold glyconanoparticles from the evaporation of a nanomolar droplet in a small spot by raster X-ray nanofluorescence. Further, raster nanocrystallography of biological objects such as rod-like tobacco mosaic virus nanoparticles reveals crystalline macro-domain formation composed of highly oriented nanorods.

Life Science Applications Utilizing Radiocarbon Tracing

Radiocarbon-based accelerator mass spectrometry (AMS) facilities at Uppsala University include a measurement center for archaeological applications and a separate entity dedicated to life science research. This paper addresses the latter, with the intention of giving a brief description of the biomedical activities at our laboratory, as well as presenting new data. The ultra-small sample preparation method, which can be used down to a few μg C samples, is outlined and complemented with new results. Furthermore, it is shown that the average secondary ion current performance for small samples can be improved by increasing the distance between the cathode surface and the pressed graphite surface. Finally, data is presented for a new application: Amyloidoses are a group of diseases where the conformational changes in specific proteins' structure lead to the formation of extracellular deposits that spread and increase in mass and eventually may lead to total organ failure and death. The formation timeframe is unknown and yet it is an important clue for the elucidation of the mechanism. We present results on bomb-peak dating of 4 different types of purified amyloid proteins from human postmortem heart and spleen samples. The data indicates that the average measured age of the carbon originating from the systemic amyloid types studied here correspond to a few years before the death of the subject. This suggests that a major part of the fibril formation takes place during the last few years before death, rather than as an accumulation of amyloid deposits over decades.

Life Science Applications Utilizing Radiocarbon Tracing

Radiocarbon-based accelerator mass spectrometry (AMS) facilities at Uppsala University include a measurement center for archaeological applications and a separate entity dedicated to life science research. This paper addresses the latter, with the intention of giving a brief description of the biomedical activities at our laboratory, as well as presenting new data. The ultra-small sample preparation method, which can be used down to a few μg C samples, is outlined and complemented with new results. Furthermore, it is shown that the average secondary ion current performance for small samples can be improved by increasing the distance between the cathode surface and the pressed graphite surface. Finally, data is presented for a new application: Amyloidoses are a group of diseases where the conformational changes in specific proteins' structure lead to the formation of extracellular deposits that spread and increase in mass and eventually may lead to total organ failure and death. The formation timeframe is unknown and yet it is an important clue for the elucidation of the mechanism. We present results on bomb-peak dating of 4 different types of purified amyloid proteins from human postmortem heart and spleen samples. The data indicates that the average measured age of the carbon originating from the systemic amyloid types studied here correspond to a few years before the death of the subject. This suggests that a major part of the fibril formation takes place during the last few years before death, rather than as an accumulation of amyloid deposits over decades.

Accelerator mass spectrometry of ultra-small samples with applications in the biosciences

An overview is presented covering the biological accelerator mass spectrometry activities at Uppsala University. The research utilizes the Uppsala University Tandem laboratory facilities, including a 5MV Pelletron tandem accelerator and two stable isotope ratio mass spectrometers. In addition, a dedicated sample preparation laboratory for biological samples with natural activity is in use, as well as another laboratory specifically for 14C-labeled samples. A variety of ongoing projects are described and presented. Examples are: (1) Ultra-small sample AMS. We routinely analyze samples with masses in the 5–10μgC range. Data is presented regarding the sample preparation method, (2) bomb peak biological dating of ultra-small samples. A long term project is presented where purified and cell-specific DNA from various part of the human body including the heart and the brain are analyzed with the aim of extracting regeneration rate of the various human cells, (3) biological dating of various human biopsies, including atherosclerosis related plaques is presented. The average built up time of the surgically removed human carotid plaques have been measured and correlated to various data including the level of insulin in the human blood, and (4) In addition to standard microdosing type measurements using small pharmaceutical drugs, pre-clinical pharmacokinetic data from a macromolecular drug candidate are discussed.

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.

Detection of nonfluorescent molecules using differential interference contrast thermal lens microscope for extended nanochannel chromatography

An ultrasensitive absorbance detector, the differential interference contrast thermal lens microscope (DIC-TLM), was employed for a chromatography system using silica nanochannel. Recently, separation of ultrasmall volume sample has been strongly required for single-cell biological and chemical analysis. Previously, we have developed a chromatography system using nanochannels of ~100 nm scale (extended nanochannels) fabricated on a silica substrate. The extended nanochromatography realized highly efficient separation of samples <1 fL without packing materials. However, its detection method was limited to fluorescence method due to the small volume, and a new detector based on absorbance has been required. On the contrary, we have also developed DIC-TLM, a photothermal spectrometer based on absorption and thermal relaxation of sample for determination of concentration of nonfluorescent molecules in extended nanochannel. In this paper, we combined the extended nanochromatography and the DIC-TLM for separation and detection of nonfluorescent dyes. Particularly, basic performances of the DIC-TLM including quantitative performance and sensitivity were deliberated for injected samples of ~fL volume.

Blank Assessment for Ultra-Small Radiocarbon Samples: Chemical Extraction and Separation Versus AMS

The Keck Carbon Cycle AMS facility at the University of California, Irvine (KCCAMS/UCI) has developed protocols for analyzing radiocarbon in samples as small as ∼0.001 mg of carbon (C). Mass-balance background corrections for modern and 14C-dead carbon contamination (MC and DC, respectively) can be assessed by measuring 14C-free and modern standards, respectively, using the same sample processing techniques that are applied to unknown samples. This approach can be validated by measuring secondary standards of similar size and 14C composition to the unknown samples. Ordinary sample processing (such as ABA or leaching pretreatment, combustion/graphitization, and handling) introduces MC contamination of ∼0.6 ± 0.3 μg C, while DC is ∼0.3 ± 0.15 μg C. Today, the laboratory routinely analyzes graphite samples as small as 0.015 mg C for external submissions and ≅0.001 mg C for internal research activities with a precision of ∼1% for ∼0.010 mg C. However, when analyzing ultra-small samples isolated by a series of complex chemical and chromatographic methods (such as individual compounds), integrated procedural blanks may be far larger and more variable than those associated with combustion/graphitization alone. In some instances, the mass ratio of these blanks to the compounds of interest may be so high that the reported 14C results are meaningless. Thus, the abundance and variability of both MC and DC contamination encountered during ultra-small sample analysis must be carefully and thoroughly evaluated. Four case studies are presented to illustrate how extraction chemistry blanks are determined.

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.

A novel radiocarbon dating technique applied to an ice core from the Alps indicating late Pleistocene ages

Ice cores retrieved from high‐altitude glaciers are important archives of past climatic and atmospheric conditions in midlatitude and tropical regions. Because of the specific flow behavior of ice, their age‐depth relationship is nonlinear, preventing the application of common dating methods such as annual layer counting in the deepest and oldest part. Here we present a new approach and technique, allowing dating of any such ice core at arbitrary depth for the age range between ∼500 years B.P. and the late Pleistocene. This new, complementary dating tool has great potential for numerous ice core related paleoclimate studies since it allows improvement and extension of existing and future chronologies. Using small to ultrasmall sample size (100 μg &gt; carbon content &gt; 5 μg) accelerator mass spectrometry, we take advantage of the ice‐included, water‐insoluble organic carbon fraction of carbonaceous aerosols for radiocarbon (14C) dating. Analysis and dating of the bottom ice of the Colle Gnifetti glacier (Swiss‐Italian Alps, 45°55′50″N, 7°52′33″E, 4455 m asl) has been successful in a first application, and the results revealed the core to cover most of the Holocene at the least with indication for late Pleistocene ice present at the very bottom.

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.