Label-free and amplified quantitation of proteins in complex mixtures using diffractive optics technology

Immunoaffinity approaches remain invaluable tools for characterization and quantitation of biopolymers. Their application in separation science is often limited due to the challenges of immunoassay development. Typical end-point immunoassays require time consuming and labor-intensive approaches for optimization. Real-time label-free analysis using diffractive optics technology (dot^®) helps guide a very effective iterative process for rapid immunoassay development. Both label-free and amplified approaches can be used throughout feasibility testing and ultimately in the final assay, providing a robust platform for biopolymer analysis over a very broad dynamic range. We demonstrate the use of dot in rapidly developing assays for quantitating (1) human IgG in complex media, (2) a fusion protein in production media and (3) protein A contamination in purified immunoglobulin preparations.     

Label-free DNA sequencing using Millikan detection

A label-free method for DNA sequencing based on the principle of the Millikan oil drop experiment was developed. This sequencing-by-synthesis approach sensed increases in bead charge as nucleotides were added by a polymerase to DNA templates attached to beads. The balance between an electrical force, which was dependent on the number of nucleotide charges on a bead, and opposing hydrodynamic drag and restoring tether forces resulted in a bead velocity that was a function of the number of nucleotides attached to the bead. The velocity of beads tethered via a polymer to a microfluidic channel and subjected to an oscillating electric field was measured using dark-field microscopy and used to determine how many nucleotides were incorporated during each sequencing-by-synthesis cycle. Increases in bead velocity of approximately 1% were reliably detected during DNA polymerization, allowing for sequencing of short DNA templates. The method could lead to a low-cost, high-throughput sequencing platform that could enable routine sequencing in medical applications.     

Label-free detection of living cervical cells based on microfluidic device with terahertz spectroscopy

Early diagnosis of cervical cancer is essential for a good prognosis. Terahertz wave detection technology is a nondestructive and label-free physical detection technology, which can detect and monitor the cancer cells in real time, especially for patients with deep or inaccessible tumors. In this study, a single-cell-layer microfluidic device was developed. After replacing the optical clearing agent, the characteristics of H8, HeLa and SiHa cell lines in adherent and suspended states were detected. Additionally, the absorption increased with increasing cell density. For the mixed suspension cell samples, principal component analysis–support vector machine method was used to identify benign and malignant cell component. After living cells formaldehyde, changes in cell membrane permeability were evaluated to identify the cell survival status (i.e., dead or living) based on terahertz spectroscopy amplitude differences. Therefore, extending the terahertz spectrum detection to the molecular level can characterize the life essence of cells and tissues.     

Label-free detection of proteins using SERS-based immuno-nanosensors

This paper describes the development and optimization of a novel class of SERS-based immuno-nanosensors for the label-free detection of specific proteins in complex environments (e.g., cell culture matrices and intracellular environments). These SERS-based nanosphere sensors are fabricated by depositing multiple layers of silver on silica nanospheres, followed by binding of the antibody of interest to the silver surface via a short rigid crosslinker. In these studies, several different crosslinkers were characterized and evaluated for optimal nanosensor activity. The crosslinkers evaluated contained either thiol or isothiocyanate functionalities, which bind to the silver surface on one end, while the other end of the crosslinker contained either a carboxylic or primary amine group, which reacts readily with the antibodies. These SERS-based nanosensors were also optimized for underlying silica sphere diameters, silica sphere coating conditions during silver deposition, number of silver layers applied, and silver surface coverage with crosslinkers. Upon optimization, the nanosensors were evaluated by monitoring their response to various antigens (e.g., human insulin or interleukin II) in complex environments.     

Label-free multiplexed virus detection using spectral reflectance imaging

We demonstrate detection of whole viruses and viral proteins with a new label-free platform based on spectral reflectance imaging. The Interferometric Reflectance Imaging Sensor (IRIS) has been shown to be capable of sensitive protein and DNA detection in a real time and high-throughput format. Vesicular stomatitis virus (VSV) was used as the target for detection as it is well-characterized for protein composition and can be modified to express viral coat proteins from other dangerous, highly pathogenic agents for surrogate detection while remaining a biosafety level 2 agent. We demonstrate specific detection of intact VSV virions achieved with surface-immobilized antibodies acting as capture probes which is confirmed using fluorescence imaging. The limit of detection is confirmed down to 3.5 × 10(5)plaque-forming units/mL (PFUs/mL). To increase specificity in a clinical scenario, both the external glycoprotein and internal viral proteins were simultaneously detected with the same antibody arrays with detergent-disrupted purified VSV and infected cell lysate solutions. Our results show sensitive and specific virus detection with a simple surface chemistry and minimal sample preparation on a quantitative label-free interferometric platform.     

Label-free virus detection using silicon photonic microring resonators

Viruses represent a continual threat to humans through a number of mechanisms, which include disease, bioterrorism, and destruction of both plant and animal food resources. Many contemporary techniques used for the detection of viruses and viral infections suffer from limitations such as the need for extensive sample preparation or the lengthy window between infection and measurable immune response, for serological methods. In order to develop a method that is fast, cost-effective, and features reduced sample preparation compared to many other virus detection methods, we report the application of silicon photonic microring resonators for the direct, label-free detection of intact viruses in both purified samples as well as in a complex, real-world analytical matrix. As a model system, we demonstrate the quantitative detection of Bean pod mottle virus, a pathogen of great agricultural importance, with a limit of detection of 10 ng/mL. By simply grinding a small amount of leaf sample in buffer with a mortar and pestle, infected leaves can be identified over a healthy control with a total analysis time of less than 45 min. Given the inherent scalability and multiplexing capability of the semiconductor-based technology, we feel that silicon photonic microring resonators are well-positioned as a promising analytical tool for a number of viral detection applications.     

Label-free detection of bacterial RNA using polydiacetylene-based biochip

A novel acetylcholinesterase (AChE)/choline oxidase (ChOx) bienzyme amperometric acetylcholine biosensor based on gold nanoparticles (AuNPs) and multi-walled carbon nanotubes (MWCNTs) has been successfully developed by self-assembly process in combination of sol-gel technique. A thiolated aqueous silica sol containing MWCNTs and ChOx was first dropped on the surface of a cleaned Pt electrode, and then AuNPs were assembled with the thiolated sol-gel network. Finally, the alternate deposition of poly (diallyldimethylammonium chloride) (PDDA) and AChE was repeated to assemble different layers of PDDA-AChE on the electrode for optimizing AChE loading. Among the resulting biosensors, the biosensor based on two layers of PDDA-AChE multilayer films showed the best performance. It exhibited a wide linear range, high sensitivity and fast amperometric response, which were 0.005-0.4mM, 3.395 μA/mM, and within 15s, respectively. The biosensor showed long-term stability and acceptable reproducibility. More importantly, this study could provide a simple and effective multienzyme immobilization platform for meeting the demand of the effective immobilization enzyme on the electrode surface.     

Digital quantification using amplified single-molecule detection

We describe a scheme for biomolecule enumeration by converting nanometer-scale specific molecular recognition events mediated by rolling-circle amplification to fluorescent micrometer-sized DNA molecules amenable to discrete optical detection. Our amplified single-molecule detection (SMD) approach preserves the discrete nature of the molecular population, allowing multiplex detection and highly precise quantification of molecules over a dynamic range of seven orders of magnitude. We apply the method for sensitive detection and quantification of the bacterial pathogen Vibrio cholerae.     

Molecular detection of cell line cross-contaminations using amplified fragment length polymorphism DNA fingerprinting technology

Summary We have tested amplified fragment length polymorphism (AFLP) technology, in comparison with isoenzyme analysis, for the simultaneous detection of inter-and intraspecific cell line cross-contaminations (CCCs) in the cell line collection held at the Istituto Zooprofilattico della Lombardia e dell’Emilia Romagna. Isoenzyme analysis identified four cases of interspecific CCCs. In a single expreiment, AFLP was able to identify the species of origin of all cell lines for which a reference genomic deoxyribonucleic acid was available and to detect five interspecific contaminations. Four CCCs confirmed data on isoenzymes, whereas the fifth CCC was detected in a species for which isoenzyme analysis was noninformative. In addition, AFLP was able to identify the putative source of the contaminations detected. The utility of the technology in the detection of intraspecific cell line contaminations, depends on the number of cell lines that have to be distinguished in a specific species and on the availability of highly informative fingerprinting systems. In mice, a single AFLP primer pair produced 16 polymorphisms and distinguished all the 15 strains of mouse cell lines analyzed. In humans, 18 AFLPs identified 83 different profiles in the 159 cell lines analyzed. Amplified fragment length polymorphism can conveniently be applied for cell line fingerprinting in species for which hypervariable markers are not available. In species for which a highly informative multiplex of microsatellite markers is available, AFLP can still provide a useful and cheap tool for simultaneously testing inter-and intraspecific contaminations.     

Label-free protein and pathogen detection using the atomic force microscope

The atomic force microscope (AFM) uses a sharp micron-scale tip to scan and amplify surface features, providing exceptionally detailed topographical information with magnification on the order of x10(6). This instrument is used extensively for quality control in the computer and semiconductor industries and is becoming a progressively more important tool in the biological sciences. Advantages of the AFM for biological application include the ability to obtain information in a direct, label-free manner and the ability to image in solution, providing real-time data acquisition under physiologically relevant conditions. A novel application of the AFM currently under development combines its surface profiling capabilities with fixed immuno-capture using antibodies immobilized in a nanoarray format. This provides a distinctive platform for direct, label-free detection and characterization of viral particles and other pathogens.     

Electrochemically amplified detection for lipopolysaccharide using ferrocenylboronic acid

A novel electrochemical technique for lipopolysaccharide (LPS) detection has been developed using a combination of ferrocenylboronic acid derivatives and an enzyme-modified electrode. The enzyme-modified electrode was constructed from a gold electrode modified with a bovine serum albumin membrane containing diaphorase. Ferrocenylboronic acid derivatives are oxidized on the electrode, and then regenerated by a diaphorase-catalyzed reaction in the presence of NADH. The consumption/regeneration cycle for ferrocenylboronic acid derivatives resulted in a chemically amplified current response. The current response for ferrocenylboronic acid derivatives decreased in association with its complexation with glycosyl units of LPS, and this current decrease caused by LPS was also amplified by the recycling process. On the other hand, the addition of a monosaccharide such as D-mannose or D-galactose induced no response at the same LPS concentration. The enzyme membrane immobilized on the electrode plays an important role in selectivity as well as chemical amplification. In addition, the enzyme-modified electrode exhibited a rapid response of 5 min for LPS, which is much faster than the currently used method. The detection limit of LPS from Escherichia coli O127:B8 was as low as 50 ng ml-1.     

Label-free and homogeneous DNA hybridization detection using gold nanoparticles-based chemiluiminescence system

We find that the catalytic activity of gold nanoparticles (GNPs) on luminol-H₂O₂ chemiluminescence (CL) system is greatly enhanced after it is aggregated by 0.5 M NaCl. We use this observation to design a CL detection of DNA hybridization. It is based on that the single- and double-stranded oligonucleotides have different propensities to adsorb on GNPs in colloidal solution, and the hybridization occurred between the probe DNA and target DNA can result in aggregation of the GNPs, producing strong CL emission. In the assay, no covalent functionalization of the GNPs, the probe, or the target DNA is required. The assay, including hybridization and detection, occurs in homogenous solution. The detection limit of target DNA (3σ) was estimated to be as low as 1.1 fM. The sensitivity was increased more than 6 orders of magnitude over that of GNPs-based colorimetric method. The present CL method for DNA hybridization detection offers the advantages of being simple, cheap, rapid and sensitive.     

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

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

Label-free detection of protein interactions using deep UV fluorescence lifetime microscopy

We present a label-free detection of protein interaction between beta-galactosidase from Escherichia coli (Ecbeta-Gal) and monoclonal anti-Ecbeta-Gal using deep UV laser-based fluorescence lifetime microscopy. The native fluorescence from intrinsic tryptophan emission was observed after one-photon excitation at 266 nm. Applying the time-correlated single-photon counting (TCSPC) method, we investigated the mean fluorescence lifetime and lifetime distributions from tryptophan residues in Ecbeta-Gal protein, monoclonal anti-Ecbeta-Gal, and corresponding complex. The results demonstrate that deep UV laser-based fluorescence lifetime microscopy is useful for sensitive identification of biological macromolecules interaction using intrinsic fluorescence.     

Label-free detection of invasive micropapillary carcinoma of the breast using multiphoton microscopy

Invasive micropapillary carcinoma of the breast (IMPC) is a rare form of breast cancer with unique histological features, and is associated with high axillary lymph node metastasis and poor clinical prognosis. Thus, IMPC should be diagnosed in time to improve the treatment and management of patients. In this study, multiphoton microscopy (MPM) is used to label-free visualize the morphological features of IMPC. Our results demonstrate that MPM images are well in agreement with hematoxylin and eosin staining and epithelial membrane antigen staining, indicating MPM is comparable to traditional histological analysis in identifying the tissue structure and cell morphology. Statistical analysis shows significant differences in the circumference and area of the glandular lumen and cancer nest between the different IMPC cell clusters with complete glandular lumen morphology, and also shows difference in collagen length, width, and orientation, indicating the invasive ability of different morphologies of IMPC may be different.     

Label-free, multiplexed detection of bacterial tmRNA using silicon photonic microring resonators

This work describes the development of an automated flow-based biosensor that employs genetically modified acetylcholinesterase (AChE) enzymes B394, B4 and wild type B131. The biosensor was based on a screen printed carbon electrode (SPE) that was integrated into a flow cell. Enzymes were immobilised on cobalt (II) phthalocyanine (CoPC) modified electrodes by entrapment in a photocrosslinkable polymer (PVA-AWP). The automated flow-based biosensor was successfully used to quantify three organophosphate pesticides (OPs) in milk samples. The OPs used were chlorpyriphos-oxon (CPO), ethyl paraoxon (EPOx) and malaoxon (MOx). The total analysis time for the assay was less than 15 min. Initially, the biosensor performance was tested in phosphate buffer solution (PBS) using B394, B131 and B4 biosensors. The best detection limits were obtained with B394; therefore, this biosensor was used to produce calibration data in milk with three OPs in the concentration range of 5 × 10(-6)M to 5 × 10(-12)M. The limit of detection (LOD) obtained in milk for CPO, EPOx and MOx were 5 × 10(-12)M, 5 × 10(-9)M and 5 × 10(-10)M, respectively, with a correlation coefficient R(2)=0.9910. The automated flow-based biosensor successfully quantified the OPs in different fat-containing milk samples. There were no false positives or false negatives observed for the analytical figures of merit for the constructed biosensors. This method is inexpensive, sensitive, portable, non-invasive and provides real-time results. This analytical system can provide rapid detection of highly toxic OPs in food matrices such as milk.     

Calibration of bioaffinity assays using kinetic data

Bioaffinity assays are usually calibrated by using a set of standard measurements fitted to a simple empirical model. In this paper, a new calibration approach based on mechanistic model of reaction kinetics is presented. When the calibration assay is known in terms of reaction mechanism, incubation time, initial concentration, and rate constants, one can back-calculate concentrations of unknown samples measured in a nonequilibrium time point. This paper describes a calculation method of unknown sample concentrations based on kinetically measured single calibration assay point. The theoretical results are verified by two common in-vitro diagnostic assays.