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A new technology platform called μSCALE combines the use of a microcapillary array with laser-based extraction to enable high-throughput biochemical and biophysical analysis and isolation of protein variants for protein-engineering applications. We describe a multipurpose technology platform, termed μSCALE (microcapillary single-cell analysis and laser extraction), that enables massively parallel, quantitative biochemical and biophysical measurements on millions of protein variants expressed from yeast or bacteria. μSCALE spatially segregates single cells within a microcapillary array, enabling repeated imaging, cell growth and protein expression. We performed high-throughput analysis of cells and their protein products using a range of fluorescent assays, including binding-affinity measurements and dynamic enzymatic assays. A precise laser-based extraction method allows rapid recovery of live clones and their genetic material from microcapillaries for further study. With μSCALE, we discovered a new antibody against a clinical cancer target, evolved a fluorescent protein biosensor and engineered an enzyme to reduce its sensitivity to its inhibitor. These protein analysis and engineering applications each have unique assay requirements and different host organisms, highlighting the flexibility and technical capabilities of the μSCALE platform.

High throughput immunoassay is increasingly crucial for both scientific and clinical applications. Here we propose a “Lab-in-a-Tip” (LIT) concept to fabricate a pipette tip containing a high-density protein array and other essential reagents. The protein array is made by self-assembling digitally encoded microparticles inside the modified tip. Mounted on a robotic workstation, it automates liquid-handling steps. Notably, compared with Luminex, the current gold standard in multiplex immunoassays, such a design enables LIT to demonstrate multiple advantages in terms of analytical sensitivity, speed, and throughput. It detects analyte concentrations as low as fg/ml, representing a sensitivity improvement of two orders of magnitude over Luminex. Incubation time is reduced to 15 minutes from Luminex’s 210 minutes. Furthermore, LIT requires only 10 µl of sample, one-fifth of what Luminex needs. This makes LIT ideal for rapid diagnostics and studies with limited biological samples, greatly expanding its application scope. High-throughput immunoassay is crucial for scientific and clinical applications. Here, the authors introduce a multiplex immunoassay platform engineered by modifying a pipette tip with a self-assembled barcoded protein array. It outperforms the gold standard in sensitivity, speed, and sample volume.

We describe highly sensitive, label-free, multiplexed electrical detection of cancer markers using silicon-nanowire field-effect devices in which distinct nanowires and surface receptors are incorporated into arrays. Protein markers were routinely detected at femtomolar concentrations with high selectivity, and simultaneous incorporation of control nanowires enabled discrimination against false positives. Nanowire arrays allowed highly selective and sensitive multiplexed detection of prostate specific antigen (PSA), PSA-α1-antichymotrypsin, carcinoembryonic antigen and mucin-1, including detection to at least 0.9 pg/ml in undiluted serum samples. In addition, nucleic acid receptors enabled real-time assays of the binding, activity and small-molecule inhibition of telomerase using unamplified extracts from as few as ten tumor cells. The capability for multiplexed real-time monitoring of protein markers and telomerase activity with high sensitivity and selectivity in clinically relevant samples opens up substantial possibilities for diagnosis and treatment of cancer and other complex diseases.

Molecular imprinting is a technique for preparing polymer scaffolds that function as synthetic receptors 1 , 2 , 3 . Imprinted polymers that can selectively bind organic compounds have proven useful in sensor development 2 , 3 , 4 , 5 , 6 , 7 . Although creating synthetic molecular-imprinting polymers that recognize proteins remains challenging 8 , 9 , 10 , 11 , nanodevices and nanomaterials show promise in this area 12 , 13 , 14 . Here, we show that arrays of carbon-nanotube tips with an imprinted non-conducting polymer coating can recognize proteins with subpicogram per litre sensitivity using electrochemical impedance spectroscopy. We have developed molecular-imprinting sensors specific for human ferritin and human papillomavirus derived E7 protein. The molecular-imprinting-based nanosensor can also discriminate between Ca 2+ -induced conformational changes in calmodulin. This ultrasensitive, label-free electrochemical detection of proteins offers an alternative to biosensors based on biomolecule recognition. Carbon nanotube tips containing imprints within a non-conducting polymer coating can detect proteins with high sensitivity, offering a label-free alternative to sensors based on biomolecule recognition.

Monitoring the kinetics of protein interactions on a high-density sensor array is vital to drug development and proteomic analysis. Label-free kinetic assays based on surface plasmon resonance are the current gold standard, but they have poor detection limits, suffer from non-specific binding, and are not amenable to high-throughput analyses. Here, we show that magnetically responsive nanosensors that have been scaled to over 100,000 sensors per cm 2 can be used to measure the binding kinetics of various proteins with high spatial and temporal resolution. We present an analytical model that describes the binding of magnetically labelled antibodies to proteins that are immobilized on the sensor surface. This model is able to quantify the kinetics of antibody–antigen binding at sensitivities as low as 20 zeptomoles of solute. Giant magnetoresistive nanosensors are used to quantify the binding kinetics of proteins at the surface of the sensor array, thus offering a sensitive assay for applications in antibody and drug development, and clinical diagnostics.

To date, the only way to array proteins with high density and high content has been to print purified proteins on a microarray surface. The next generation of nucleic acid programmable protein arrays (NAPPA) now allows thousands of proteins to be produced in situ on a microarray. We developed a high-density self-assembling protein microarray, based on the nucleic acid programmable protein array (NAPPA) concept, to display thousands of proteins that are produced and captured in situ from immobilized cDNA templates. We arrayed up to 1,000 unique human cDNAs and obtained high yields of protein expression and capture with minimal variation and good reproducibility. This method will enable various experimental approaches to study protein function in high throughput.

Protein microarrays provide a powerful tool for the study of protein function. However, they are not widely used, in part because of the challenges in producing proteins to spot on the arrays. We generated protein microarrays by printing complementary DNAs onto glass slides and then translating target proteins with mammalian reticulocyte lysate. Epitope tags fused to the proteins allowed them to be immobilized in situ. This obviated the need to purify proteins, avoided protein stability problems during storage, and captured sufficient protein for functional studies. We used the technology to map pairwise interactions among 29 human DNA replication initiation proteins, recapitulate the regulation of Cdt1 binding to select replication proteins, and map its geminin-binding domain.

ABSTRACT Microarrays with biomolecules (e.g., DNA and proteins), cells, and tissues immobilized on solid substrates are important tools for biological research, including genomics, proteomics, and cell analysis. In this paper, the current state of microarray fabrication is reviewed. According to spot formation techniques, methods are categorized as \"contact printing\" and \"non-contact printing.\" Contact printing is a widely used technology, comprising methods such as contact pin printing and microstamping. These methods have many advantages, including reproducibility of printed spots and facile maintenance, as well as drawbacks, including low-throughput fabrication of arrays. Non-contact printing techniques are newer and more varied, comprising photochemistry-based methods, laser writing, electrospray deposition, and inkjet technologies. These technologies emerged from other applications and have the potential to increase microarray fabrication throughput; however, there are several challenges in applying them to microarray fabrication, including interference from satellite drops and biomolecule denaturization.

There is an increasing awareness that health care must move from post-symptomatic treatment to presymptomatic intervention. An ideal system would allow regular inexpensive monitoring of health status using circulating antibodies to report on health fluctuations. Recently, we demonstrated that peptide microarrays can do this through antibody signatures (immunosignatures). Unfortunately, printed microarrays are not scalable. Here we demonstrate a platform based on fabricating microarrays (~10 M peptides per slide, 330,000 peptides per assay) on silicon wafers using equipment common to semiconductor manufacturing. The potential of these microarrays for comprehensive health monitoring is verified through the simultaneous detection and classification of six different infectious diseases and six different cancers. Besides diagnostics, these high-density peptide chips have numerous other applications both in health care and elsewhere. Health monitoring based on measuring circulating antibodies may enable the presymptomatic detection of diseases. Here, the authors report a large-scale peptide array platform that allows for a detection of the profile of circulating antibodies associated with cancers and infectious diseases.

Monoclonal antibodies that recognize specific antigens of interest are used as therapeutic agents and as tools for biomedical research 1 . Discovering a single monoclonal antibody requires retrieval of an individual hybridoma from polyclonal mixtures of cells producing antibodies with a variety of specificities. The time required to isolate hybridomas by a limiting serial-dilution, however, has restricted the diversity and breadth of available antibodies. Here we present a soft lithographic method based on intaglio printing to generate microarrays comprising the secreted products of single cells. These engraved arrays enable a rapid (<12 h) and high-throughput (>100,000 individual cells) system for identification, recovery and clonal expansion of cells producing antigen-specific antibodies. This method can be adapted, in principle, to detect any secreted product in a multiplexed manner.