A vertical microfluidic probe

Performing localized chemical events on surfaces is critical for numerous applications. We earlier invented the microfluidic probe (MFP), which circumvented the need to process samples in closed microchannels by hydrodynamically confining liquids that performed chemistries on surfaces (Juncker et al. Nat. Mater. 2005, 4, 622-628). Here we present a new and versatile probe, the vertical MFP (vMFP), which operates in the scanning mode while overcoming earlier challenges that limited the practical implementation of the MFP technology. The key component of the vMFP is the head, a microfluidic device (～1 cm(2) in area) consisting of glass and Si and having microfluidic features fabricated in-plane in the Si layer. The base configuration of the head has two micrometer-size channels that inject/aspirate liquids and terminate at the apex which is ～1 mm(2). In scanning mode, the head is oriented vertically with the apex parallel to the surface with typical spacing of 1-30 μm. Such length scales and using flow rates from nanoliters/second to microliters/second allow chemical events to be performed on surfaces with tens of picoliter quantities of reagents. Before scanning, the head is clipped on a holder for leak-free, low dead volume interface assembly, providing a simple world-to-chip interface. Surfaces are scanned by mounting the holder on a computer-controlled stage having ～0.1 μm resolution in positioning. We present detailed steps to fabricate vMFP heads having channels with dimensions from 1 μm × 1 μm to 50 μm × 50 μm for liquid localization over areas of 10-10,000 μm(2). Additionally, advanced design strategies are described to achieve high yield in fabrication and to support a broad range of applications. These include particulate filters, redundant aperture architectures, inclined flow-paths that service apertures, and multiple channels to enable symmetric flow confinement. We also present a method to characterize flow confinement and estimate the distance between the head and the surface by monitoring the evolution of a solution of fluorescently labeled antibody on an activated glass surface. This flow characterization reveals regimes of operation suitable for different surface topographies. We further integrate the dispensing of immersion liquid to the vMFP head for processing surfaces for extended periods of time (～60 min). The versatility of the vMFP is exemplified by patterning fluorescently labeled proteins, inactivation of cells using sodium hypochlorite, and staining living NIH fibroblasts with Cellomics. These applications are enabled by the compact design of the head, which provides easy access to the surface, simplifies alignment, and enables processing surfaces having dimensions from the micrometer to the centimeter scale and with large topographical variations. We therefore believe that ease-of-operation, reconfigurability, and conservative use of chemicals by the vMFP will lead to its widespread use by microtechnologists and the chemical and biomedical communities.     

A technique for relative quantitation of cancer biomarkers in formalin‐fixed,paraffin‐embedded (FFPE) tissue using stable‐isotope‐label based mass spectrometry imaging (SILMSI)

We developed a novel technique for the relative quantitation of pairs of cancer biomarkers in formalin‐fixed paraffin‐embedded (FFPE) tissue. The method utilizes stable isotope labeled (SIL) chromogens deposited during the standard immunohistochemistry (IHC) tissue staining process. The labeled chromogens are precipitated on tissue enzymatically using the standard IHC protocols. The tissue is then imaged with matrix‐free laser desorption ionization time‐of‐flight mass spectrometry, and peak intensities of reporter ions are used to estimate the relative quantitation of protein biomarkers across the tissue. The relative abundance of two breast cancer biomarkers, estrogen receptor (ER) and progesterone receptor (PgR), were quantitated using their ratio of expression in xenograft models, and the ratios were found to be reproducible both within and across serial sections. The relative quantification of multiple biomarkers in situ across a single tissue section adds an additional dimension in cancer histological evaluation by allowing a visual and statistical assessment of tumor heterogeneity. Copyright © 2015 John Wiley & Sons, Ltd.     

Imaging mass spectrometry of gold nanoparticles in a tissue section as an immunohistochemical staining mass probe

For analysis of low abundance peptides in a tissue section, immunohistochemical staining through antibody‐antigen interaction is a usual technique. The antibody is conjugated with a probe moiety that aids in highly sensitive detection. Gold nanoparticles, which show excellent chemical stability and variation of surface modifications, are expected to act as a sensitive mass probe to desorb gold ions (Au⁺, Au₂⁺, Au₃⁺) that are distinguishable from fragment ions from organic molecules. Here, green fluorescent proteins (GFP) in a tissue section of a transgenic zebrafish were detected by the gold mass probe conjugated with antibodies. Due to the efficient ionization and desorption of gold ions, imaging mass spectrometry of Au₂⁺ ions indicated the distribution of gold nanoparticles stained in a tissue section, and the mass signal distribution was consistent with the area where the GFP‐expressing cells were distributed. Conventional immunofluorescence techniques showed intense autofluorescence that come from intrinsic fluorophores in the tissue section. In contrast, the gold nanoparticles acted as an immunostaining mass probe that displayed significantly lower background signals.     

Immunochemical detection of peptides and proteins on press-blots after direct tissue gel isoelectric focusing

A sensitive method is described for the detection of tissue peptides and proteins. They are separated by tissue isoelectric focusing using thin large-pore polyacrylamide gels, containing detergent and dimethylformamide, and are fixed with either glutaraldehyde or formaldehyde in gelatin-coated nitrocellulose membranes using press-blotting. The fixed peptide and protein antigens are visualized by immunoperoxidase staining. The spectrum of fixed tissue constituents may also be used to test antiserum reactivity and specificity in immunocytochemical staining procedures. Isoelectric focusing of 2 microL homogenates of the neurointermediate lobe of the pituitary allowed the immunodetection of peptides and proteins of various sizes and the determination of isoelectric points. However, direct application onto gels of small pieces of frozen tissue sections, sliced in a cryostat, appeared to be more efficient. By direct tissue isoelectric focusing of brain tissue, peptides were effectively eluted and separated from sections up to 100 microns thickness. This allowed the detection of small peptides with a detection limit of approximately 10 pg/section.     

Review of analytical measurements facilitated by drop formation technology

The use of drops in chemical analysis methodology and instrumentation has a deeply rooted past in the area of electrochemistry through the evolution of the dropping mercury electrode (DME). This history has also been deeply rooted in the field of surface science due to the inextricable connection between surface tension forces and drop formation. While the use of the DME is well established, the evolution of drop-based analytical measurements using aqueous and/or organic drops is a rapidly emerging and diverse field, encompassing several interdisciplinary areas of science: surface science and interfacial surface tension phenomena, spectroscopic detection, analytical instrumentation hyphenation, liquid membrane separation, reagent chemistry, electrochemistry, and so on. This review of 112 references covers various aspects of drop-based analytical measurements involving aqueous and/or organic drops. The review is divided into four sections, although the classification of a particular reference into a given section can sometimes be argued. The first section considers the use of drops as a detector component. The second section deals with fundamental studies that probe drop-related chemical and physical phenomena that are relevant to current and future developments in analytical chemistry. The next section covers recent advances in the area of microfluidic sample handling and instrumentation hyphenation. The final section reports upon emerging technologies aimed toward drop-based chemical analyzers that incorporate a number of steps in a chemical analysis: microextraction, preconcentration, reagent chemistry, microfluidic handling, and detection.     

Microspot-based ELISA in microfluidics: chemiluminescence and colorimetry detection using integrated thin-film hydrogenated amorphous silicon photodiodes

Microfluidic technology has the potential to decrease the time of analysis and the quantity of sample and reactants required in immunoassays, together with the potential of achieving high sensitivity, multiplexing, and portability. A lab-on-a-chip system was developed and optimized using optical and fluorescence microscopy. Primary antibodies are adsorbed onto the walls of a PDMS-based microchannel via microspotting. This probe antibody is then recognised using secondary FITC or HRP labelled antibodies responsible for providing fluorescence or chemiluminescent and colorimetric signals, respectively. The system incorporated a micron-sized thin-film hydrogenated amorphous silicon photodiode microfabricated on a glass substrate. The primary antibody spots in the PDMS-based microfluidic were precisely aligned with the photodiodes for the direct detection of the antibody-antigen molecular recognition reactions using chemiluminescence and colorimetry. The immunoassay takes ~30 min from assay to the integrated detection. The conditions for probe antibody microspotting and for the flow-through ELISA analysis in the microfluidic format with integrated detection were defined using antibody solutions with concentrations in the nM-μM range. Sequential colorimetric or chemiluminescence detection of specific antibody-antigen molecular recognition was quantitatively detected using the photodiode. Primary antibody surface densities down to 0.182 pmol cm(-2) were detected. Multiplex detection using different microspotted primary antibodies was demonstrated.     

High spatial resolution imaging mass spectrometry and classical histology on a single tissue section

The work presented in this report describes and demonstrates a protocol for protein imaging analysis of biological tissue using MALDI IMS where histological staining and MS analysis are performed on the same tissue section. Spatial image resolution is shown at 35 μm for sagittal sections of the cerebellum from rat brain.     

HistoFlex--a microfluidic device providing uniform flow conditions enabling highly sensitive, reproducible and quantitative in situ hybridizations

A microfluidic device (the HistoFlex) designed to perform and monitor molecular biological assays under dynamic flow conditions on microscope slide-substrates, with special emphasis on analyzing histological tissue sections, is presented. Microscope slides were reversibly sealed onto a cast polydimethylsiloxane (PDMS) insert, patterned with distribution channels and reaction chambers. Topology optimization was used to design reaction chambers with uniform flow conditions. The HistoFlex provided uniform hybridization conditions, across the reaction chamber, as determined by hybridization to microscope slides of spotted DNA microarrays when applying probe concentrations generally used in in situ hybridization (ISH) assays. The HistoFlex's novel ability in online monitoring of an in situ hybridization assay was demonstrated using direct fluorescent detection of hybridization to 18S rRNA. Tissue sections were not visually damaged during assaying, which enabled adapting a complete ISH assay for detection of microRNAs (miRNA). The effects of flow based incubations on hybridization, antibody incubation and Tyramide Signal Amplification (TSA) steps were investigated upon adapting the ISH assay for performing in the HistoFlex. The hybridization step was significantly enhanced using flow based incubations due to improved hybridization efficiency. The HistoFlex device enabled a fast miRNA ISH assay (3 hours) which provided higher hybridization signal intensity compared to using conventional techniques (5 h 40 min). We further demonstrate that the improved hybridization efficiency using the HistoFlex permits more complex assays e.g. those comprising sequential hybridization and detection of two miRNAs to be performed with significantly increased sensitivity. The HistoFlex provides a new histological analysis platform that will allow multiple and sequential assays to be performed under their individual optimum assay conditions. Images can subsequently be recorded either in combination or sequentially through the ability of the HistoFlex to monitor assays without disassembly.     

Liquid microjunction surface sampling probe electrospray mass spectrometry for detection of drugs and metabolites in thin tissue sections

A self-aspirating, liquid microjunction surface sampling probe/electrospray emitter mass spectrometry system was demonstrated for use in the direct analysis of spotted and dosed drugs and their metabolites in thin tissue sections. Proof-of-principle sampling and analysis directly from tissue without the need for sample preparation was demonstrated first by raster scanning a region on a section of rat liver onto which reserpine was spotted. The mass spectral signal from selected reaction monitoring was used to develop a chemical image of the spotted drug on the tissue. The probe was also used to selectively spot sample areas of sagittal whole-body tissue from a mouse that had been dosed orally (90 mg/kg) with R,S-sulforaphane 3 h prior to sacrifice. Sulforaphane and its glutathione and N-acetyl cysteine conjugates were monitored with selected reaction monitoring and detected in the stomach and various other tissues from the dosed mouse. No signal for these species was observed in the tissue from a control mouse. The same dosed-tissue section was used to illustrate the possibility of obtaining a lane scan across the whole-body section. In total, these results illustrate the potential for rapid screening of the distribution of drugs and metabolites in thin tissue sections with the liquid micro-junction surface sampling probe/electrospray mass spectrometry approach. Published in 2007 by John Wiley & Sons, Ltd.     

An Integrated Microfluidic Probe for Mass Spectrometry Imaging of Biological Samples**

Ambient ionization based on liquid extraction is widely used in mass spectrometry imaging (MSI) of molecules in biological samples. The development of nanospray desorption electrospray ionization (nano-DESI) has enabled the robust imaging of tissue sections with high spatial resolution. However, the fabrication of the nano-DESI probe is challenging, which limits its dissemination to the broader scientific community. Herein, we describe the design and performance of an integrated microfluidic probe (iMFP) for nano-DESI MSI. The glass iMFP, fabricated using photolithography, wet etching, and polishing, shows comparable performance to the capillary-based nano-DESI MSI in terms of stability and sensitivity; a spatial resolution of better than 25 μm was obtained in these first proof-of-principle experiments. The iMFP is easy to operate and align in front of a mass spectrometer, which will facilitate broader use of liquid-extraction-based MSI in biological research, drug discovery, and clinical studies.     

Proteomic analysis of immunostained,laser-capture microdissected brain samples

Proteomic analysis is often performed on homogenized preparations of whole tissues, which does not provide any information about relevant biochemical changes in specific cell types. Laser-capture microdissection (LCM) is a technique that is precise enough to dissect single cells within a tissue section. Phenotypically defined cells of interest may be visualized by immunostaining prior to microdissection. Previously published immunostaining protocols adapted to LCM require the use of very high antibody titers and very short incubation times. This raises the concern that low-abundance antigens would not be detected and that antisera would be rapidly depleted. In addition, protein recovery from samples was not evaluated in most of these studies. Here, we describe an optimized immunostaining method based on immunofluorescence. By comparing two-dimensional electrophoresis (2-DE) results obtained from immunostained LCM brain tissue samples to those obtained from unstained, manually dissected samples, we demonstrated that immunofluorescent staining gave comparable protein recovery and similar resolution of protein spots on 2-DE gels. Moreover, matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry analysis of selected spots from gels derived from control and immunostained LCM samples revealed that the immunostaining process had minimal effect on protein identification. LCM of immunofluorescently labeled tissue sections is a practical and powerful method to perform proteomic studies on specifically defined cell groups.     

Screen-printed microfluidic device for electrochemical immunoassay

In this paper, a new microfluidic array device has been fabricated with screen printing technology. In contrast to traditional microfabrication processes, our method is simple, inexpensive and also suitable for mass production. The device is used for sandwich-type electrochemical immunoassay, in which probes are covalently attached to the electrode surface via electropolymerized polypyrrole propylic acid (PPA) film. This novel microfluidic system enables the whole array preparation and detection processes, including the probe immobilization, sample injection, enzyme incubation and electrochemical detection, to be conducted in the sealed microchannels. For a demonstration, mouse IgG is selected as the target analyte and its detection is realized by sandwich ELISA with goat anti-mouse IgG, rat anti-mouse IgG (conjugated to alkaline phosphatase) and p-aminophenyl phosphate (PAPP) as the primary antibody, second antibody, and enzyme substrate, respectively. A detection limit of 10 ng mL(-1) (67 pM) is achieved with a dynamic range of 100 ng mL(-1)-10 microg mL(-1). In addition, anti-goat IgG is also immobilized as an alternative probe to test mouse IgG in the solution, in order to demonstrate the multiplexing capability as well as the specificity of the device. As expected, the electrochemical responses are much lower than that using anti-mouse IgG as the probe, indicating good selectivity of the immunoassay device. These results indicate a great promise toward the development of miniaturized, low-cost protein biochips for clinical, forensics, environmental, and pharmaceutical applications.     

High-resolution,imaging TOF-SIMS: novel applications in medical research

The use of high-resolution, imaging TOF-SIMS is described and examples are made to demonstrate the application of the method in medical research. Cytochemistry by TOF-SIMS is shown by localization of diacylglycerol (DG) in cryostat sections of hyaline cartilage and by localization of corticosterone in cryostat sections of the adrenal gland cortex. Quantitative measurements and comparison of groups is shown by comparing the lipid content of adipose tissue from two mouse strains, transgenic mouse expressing the FOXC2 gene and wild-type controls. Finally, biopsies made for histopathological diagnosis of infantile reversible cytochrome c oxidase deficiency myopathy were analyzed in order to define the chemical content of areas showing a pathological structure in the light microscope. The use of high-resolution, imaging TOF-SIMS in medical research allows analysis of intact tissue and probe-free localization of specific target molecules in cells and tissues. The TOF-SIMS analysis is not dependent on penetration of reagents into the sample and also independent of probe reactivity such as cross-reactivity or background staining. The TOF-SIMS method can be made quantitative and allows for analysis of specific target molecules in defined tissue compartments.     

敞开式质谱成像方法的组织样品稳定性研究

以添加镇静催眠候选新药N6-羟苄腺嘌呤核苷[N6-(4-hydroxybenzyl)adenine riboside,NHBA]的组织匀浆切片作为考察对象,对空气动力辅助离子化质谱成像系统(Air Flow Assisted Ionization Mass Spectrometry Imaging,AFAI-MSI)的关键参数进行考察与优化,以保证最佳条件进行样品检测。在此基础上,对预处理的整体大鼠组织切片进行平行的连续两次质谱成像分析,考察了其内源性代谢物在成像分析过程中是否发生变化。通过对采集数据中关键质谱峰的筛选等处理步骤,并采用可视化的主成分分析(PCA)方法,开展了组织样品内源性代谢物的稳定性分析,最终验证了采用该样品前处理和质谱成像方法,能够保证组织切片样品中内源性代谢物的稳定性,为质谱成像分析结果提供了可靠依据。     

Integrated microfluidic bioprocessor for solid phase capture immunoassays

A microfluidic device for solid-phase immunoassays based on microparticle labeling is developed using microvalve-control structures for automated sample processing. Programmable microvalve control in a multilayer structure provides automated sample delivery, adjustable hydrodynamic washing and compatibility with a wide range of substrates. Capture antibodies are derivatized on glass surfaces within the processor using an APTES patterning method, and magnetic microspheres conjugated with a secondary detection antibody are used as labels in a capture-sandwich format. In this microfluidic processor, washing force can be precisely controlled to remove the nonspecifically bound microparticles. Automated microfluidic immunoassays are demonstrated for mouse immunoglobulin (IgG) and human prostate specific antigen (PSA) with limits of detection of 1.8 and 3 pM, respectively. The sample processor architecture is easily parallelized for high-throughput analysis and easily interfaced with various assay substrates.     

Direct immunoblotting from histological sections of brain onto nitrocellulose: evaluation with the protein neurophysin

A method for the isolation and localization of proteins and peptides from histological sections of rat and human brain by immunoblotting is described. For validation, the well-characterized protein neurophysin was electrophoretically transferred from formaldehyde-fixed or fresh tissue sections onto a nitrocellulose membrane. Neurophysin on the nitrocellulose membrane was detected by a specific antibody reaction. The antibody against neurophysin was visualized either by using secondary antibodies, conjugated with peroxidase or by protein A gold, followed by enhancement with silver. With this simple and fast method, neurophysin (or other proteins and peptides) can be identified on nitrocellulose membranes in areas that correspond to anatomically defined regions. Since the procedure combines the advantages of precise regional localization of polypeptides with the specificity of antibody-antigen reactions, the method may prove useful for rapid screening of the distribution of peptides or proteins in (brain) tissue.     

Distribution of olfactory marker protein on a tissue section of vomeronasal organ measured by AFM

Distribution of olfactory marker protein (OMP) on a tissue section of vomeronasal organ (VNO) was successfully measured by atomic force microscopy (AFM). Anti-OMP antibodies were covalently crosslinked with the tip of the AFM and were used as a probe to observe the distribution of OMP on a tissue section. First, force measurements were performed using a glass surface on which OMP was covalently immobilized to verify the success of tip modification. Clear differences of interaction forces were observed between a specific pair and the control experiments, indicating that the tip preparation succeeded. Next, distributions of OMP on the tissue section were observed by AFM and were compared with immunohistochemical observations. For large scale observation, a microbead was used as a probe in the AFM measurements. The results of the AFM measurements were well overlapped with that of immunohistochemistry, confirming the reliability of our method. A mapping of the AFM measurement with high resolution was also successfully obtained, which showed an advantage of the application of the AFM measurement in analysis of proteins on the tissue section.     

Polyurea microcapsules: Surface modification and capsule size control

A postmodification method for polyurea microcapsule (PUMC) surfaces using functional polyelectrolytes is reported in this article. Fluorescein isothiocyanate (FITC) was used to probe the chemistry on PUMC surface and label nucleophilic groups on the surface, in particular amines. As well, a fluorescently labeled polyanion containing electrophilic acetoacetate groups was used to covalently react with these nucleophilic groups on the PUMC surfaces. This modification causes charge reversion of the originally cationic PUMC and enables subsequent layer‐by‐layer (LbL) coating using other polyelectrolytes, allowing for covalent or noncovalent modification of the capsule surface. All modification steps were monitored using either laser scanning confocal microscopy or fluorescence microscopy. Optical and fluorescence microscopy of PUMC wall cross‐sections embedded in resin confirmed that the modifications were restricted to the outer surface of PUMCs, offering minimum interference of this modification method with other capsule wall properties. In addition, a simple T‐junction type microfluidic device based on a commercially available MicroTEE was designed to produce narrowdisperse PUMCs. This device was easy to set up and operate and was proved to be an useful tool for making monodisperse emulsions and narrowdisperse MCs. © 2011 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2011     

Numerical simulation of polymerization in interdigital multilamination micromixers

Free radical polymerization in microfluidic devices modeled with the help of numerical simulations is discussed. The simulation method used allows the simultaneous solvation of partial differential equations resulting from the hydrodynamics, thermal and mass transfer (convection, diffusion and chemical reaction). Three microfluidic devices are modeled, two interdigital multilamination micromixers respectively with a large and short focusing section, and a simple T-junction followed by a microtube reactor together considered as a bilamination micromixer with a large focusing section. The simulations show that in spite of the heat released by the polymerization reaction, the thermal transfer in such microfluidic devices is high enough to ensure isothermal conditions. Moreover, for low radial Peclet number, microfluidic devices with a large focusing section can achieve better control over the polymerization than a laboratory scale reactor as the polydispersity index obtained is very close to the theoretical limiting value. As the characteristic dimension of the microfluidic device increases, i.e. for high radial Peclet number, the reactive medium cannot be fully homogenized by the diffusion transport before leaving the system resulting in a high polydispersity index and a loss in the control of the polymerization.     

Multiple functions of microfluidic platforms: Characterization and applications in tissue engineering and diagnosis of cancer

Microfluidic system, or lab-on-a-chip, has grown explosively. This system has been used in research for the first time and then entered in the clinical section. Due to economic reasons, this technique has been used for screening of laboratory and clinical indices. The microfluidic system solves some difficulties accompanied by clinical and biological applications. In this review, the interpretation and analysis of some recent developments in microfluidic systems in biomedical applications with more emphasis on tissue engineering and cancer will be discussed. Moreover, we try to discuss the features and functions of microfluidic systems.