This paper describes the use of focused electric fields and focused optical fields for the high-resolution manipulation of single cells. A focused electric field, obtained with the use of ultramicroelectrodes (tip diameter approximately 5 μm), is used to electroporate and electrofuse individual cells selectively and with high spatial resolution. A focused optical field, in the form of an optical tweezer, is used to isolate single organelles from a cell as well as to position liposomes incorporated with receptors and transporters along the cell for the high-resolution sampling and probing of the cellular microenvironment.
Tuberculosis (TB) remains the leading cause of death in the world from a single infectious disease, and the threat is becoming more critical with the spread of multi-drug resistant Tuberculosis (MDR-TB). TB detection, and susceptibility testing for drug resistant strain identification, is advancing with the development of Luciferase Reporter Mycobacteriophages (LRM). LRM will emit visible light at very low intensity when in the presence of live mycobacteria cells such as Tuberculosis strains. InterScience, Inc., together with its collaboration, is developing a highly sensitive, real-time digital detection system for the analysis of luminescent assays. Recent advances in system sensitivity, design, and implementation, as well as preliminary results of the development of individual test cartridges, will be presented. The ultimate goal of this work is to provide a versatile luminescence detection tool for widespread research and clinical applications.
A number of theoretical and practical limits of high-speed flow cytometry/cell sorting are important for clinical diagnostics and therapeutics. Three applications include: (1) stem cell isolation with tumor purging for minimal residual disease monitoring and treatment, (2) identification and isolation of human fetal cells from maternal blood for prenatal diagnostics and in-vitro therapeutics, and (3) high-speed library screening for recombinant vaccine production against unknown pathogens.
We have developed a new fluorescence polarization (FP) reader suitable for high-throughput screening (HST) and ultra-HTS whose assay-performance and sample-throughput are both considerably improved over present state-of-the-art instrumentation. The SymmetryTM reader possesses a number of features that differ from conventional HTS FP readers. These include: laser-based excitation, liquid crystal polarization optics that rapidly and accurately measure polarization states; and CCD detectors to capture emission from multiple wells. We show that the performance in assays relevant to the drug discovery process, such as G- protein coupled receptor-based assays, is significantly enhanced due to a dramatic improvement in precision. Furthermore, the CCD-detection system used can substantially improve sample throughput compared to sequential readers while maintaining high performance.
The monitoring of various tissue's physiological and biochemical parameters is one of the tools used by the clinicians to improve diagnosis capacity. As of today, the very few devices developed for real time clinical monitoring of tissue vitality are based on a single parameter measurement. Tissue energy balance could be defined as the ratio between oxygen or energy supply and demand. In order to determine the vitality of the brain, for example, it is necessary to measure at least the following 3 parameters: Energy Demand--potassium ion homeostasis; Energy Supply-- cerebral blood flow; Energy Balance--mitochondrial NADH redox state. For other tissues one can measure various energy demand processes specific to the tested organ. We have developed a unique multiparametric monitoring system tested in various experimental and clinical applications. The multiprobe assembly (MPA) consists of a fiber optic probe for measurement of tissue blood flow and mitochondrial NADH redox state, ion selective electrodes (K+, Ca2+, H+), electrodes for electrical activities (ECoG or ECG and DC potential), temperature probe and for monitoring the brain - Intra Cranial Pressure probe (ICP). The computerized monitoring system was used in the neurological intensive care unit to monitor comatose patients for a period of 24-48 hours. Also, a simplified MPA was used in the neurosurgical operating room or during organ transplantation procedure. It was found that the MPA could be used in clinical situations and that the data collected has a significant diagnosis value for the medical team.
An automated biosensor for 1-hydroxypyrene-glucuronide (OHPG) has been developed using a sensor platform initially designed for aflatoxin. The platform is based on the properties of immunoaffinity for sample purification and concentration, and fluorescence for detection. Experiments have demonstrated capture, wash, elution, and quantitation with very good results. The device is handheld and battery- driven, and can be miniaturized further. The bed volume of the column was 0.1 ml with a capacity of about 400 ng of the OHPG. Analysis time was 10 - 11 minutes. The sensitivity was about 0.5 ppb. The antibodies for the system were developed previously and recognize OHPG. The biosensor relies upon microprocessor-controlled minifluidics and fluorometry.
A simple and inexpensive colorimetric spectrometer for determining total cholesterol has been developed, comprising a diode laser as the light source, optical fibers for the light guide and a photodiode as the detector. In order to generate the color change, enzymatic reaction of cholesterol oxidase-peroxidase method was used. The stability and performance of the new system was investigated by obtaining the calibration curve for standard cholesterol solutions. The total cholesterol in human serum was also measured by the analyzer and compared with the value obtained by a conventional chemistry analyzer. The results showed that the developed spectrometer was useful for the determination of cholesterol levels.
A biosensor based on long period grating (LPG) technology has been used to demonstrate the detection of large molecules (proteins) and small molecules (pesticides). The LPG sensor is a spectral loss optical fiber based system that provides direct detection of large molecules, by using an antigen or antibody modified hydrogel, without the need for secondary amplification. The binding of the specific target results in a mass increase that produces a localized refractive index change around the LPG region and thus a spectral shift in the observed wavelength loss band. The magnitude of the observed shift can be correlated to target concentration. The HIV protein p24 was directly detected at 1 ng/mL with a specific signal that was 5 - 7 times that of the system noise. A direct and indirect competitive assay was demonstrated with the target atrazine. The sensitivity of the two competitive assay formats was in the range of 10 - 50 ng/mL. In all three-assay examples, the biosensor was regenerated by treatment with 50 mM HCl and reused. The LPG biosensor offers speed (results in less than five minutes), versatility, reuse, specificity and sensitivity.
Great new product ideas come from many sources including academia, industry and creative entrepreneurs. Combine a solid team of scientists, engineers and business people with diverse, relevant experience with committed investors and those ideas can become reality. The development of the LifeLiteTM System is a case study that began with an academic research program and will culminate in commercial launch in IIQ01 following FDA approval.
The LifeLiteTM System is a technology platform with broad application in immunoassays, molecular diagnostics and genomics/proteomics. The first product application is the LifeLiteTM Cardiac Panel, a 5-minute point-of-care test to measure troponin-I, myoglobin and CK-MB in whole blood using a single disposable reagent cartridge. Each cartridge also contains proprietary integral quality controls to check that the instrument and reagents are functioning propertly on every test. No other system offers the superior performance, single cartridge/multi-analyte testing capability and breadth of new product candidates.
This paper describes some of the key technical challenges and creative solutions applied by the ThauMDx product development team to apply EPWTM in a commercial product as well as future applications of the platforms.
Principal component analysis (PCA) in the wavelet domain provides powerful new features for the non-invasive detection of cervical intraepithelial neoplasia (CIN) using fluorescence imaging spectroscopy. These features are known as principal wavelet components (PWCs). The multiscale structure of the fluorescence spectrum for each pixel of the hyperspectral data cube is extracted using the continuous wavelet transform. PCA is then used to compress and denoise the wavelet representation for presentation to a feed- forward neural network for tissue classification. Using PWC features as inputs to a 5-class NN resulted in average correct classification rates of 95% over five cervical tissue classes corresponding to low-grade dysplasia, squamous, columnar, metaplasia plus a fifth class for other unspecified tissue types, blood and mucus. A 2-class NN was also trained to discriminate between CIN1 and normal tissue with sensitivity and specificity of 98% and 99%, respectively. All performance assessments were based on test data from a set of patients not seen during NN training. Trained neural classifiers were used to `compress' and transform 3D hyperspectral data cubes into 2D color-coded images that accurately mapped the spatial distribution of both normal and dysplastic tissue over the surface of the entire cervix.
A transmittance pulse oximetry system based on near-infrared laser diodes (LD) for monitoring arterial blood hemoglobin oxygen saturation (So2) has been previously reported. In this work we present the results obtained after improvements in the sensor configuration, signal processing algorithm and calibration procedure. The pulse oximetry system also comprises the sensor electronics, and a data acquisition board installed on a handheld personal computer. The two LD chips are mounted on a single metal heat-sink and as photo- detectors are used silicon p-i-n photodiodes with the first amplifier stage situated in their back side. The real time calculation of the parameters related to So2 is carried out through a numeric separation of the pulsatile and non- pulsatile components of the photoplethysmographic signals for both wavelengths and a non-linear filtering. Patients with respiratory failure conditions were monitored as a part of the calibration procedure in order to cover a wide range of So2-values. A calibration curve have been derived through the determination of in vitro arterial So2 with a significant quantity of experimental points ranging from 60 to almost 100%. The obtained results demonstrate that it is possible to apply the proposed system to monitoring a wide range of oxygen saturation levels.
Recently, the function of zinc in the axonal boutons of hippocampal neurons has come under increased scrutiny as evidence has emerged of a putative role for this metal ion in neural damage following insults such as ischemia, blunt force trauma, and seizure. Indeed, the nonpathological role of free zinc in the brain remains cryptic after more than 40 years. We have used a biosensing approach to determine free zinc ion concentrations by fluorescence lifetime, intensity, intensity ratio, or anisotropy changes caused by binding of zinc to variants of a protein, apocarbonic anhydrase II (apo-CA). This approach permits real time measurement of zinc down to picomolar levels, with no perceptible interference from other divalent metal ions abundant in serum and tissue, such as calcium and magnesium. Recently, we used apo-CA together with a fluorescent ligand whose binding is metal-dependent to obtain the first fluorescence micrographs of zinc release from a rat hippocampus model in response to electrical stimulus. In our view, elucidation of the zinc fluxes in neural tissue ultimately requires quantitation, as in the case of calcium. Recent results will be shown.
We have developed new, fluorescence polarization-based approaches for performing enzyme assays in homogeneous solutions and for detecting the hybridization of peptide nucleic acids to DNA targets.
In the first method, fluorescein-labeled peptides serving as protein kinase sustrates are thiophosphorylated in the presence of the ATP analog ATPγS. A sulfer-reactive biotin derivative is then added to the mixture and allowed to react with the thiophosphorylated peptide. The formation of a fluorescein-labeled, biotinylated product can be detected by measuring the fluorescence polarization signal of fluorescein upon addition of streptavidin.
In the second method, fluorescein-labeled peptides are subjected to enzymatic phosphorylation, desphosphorlation, or proteollytic cleavage by protein kinases, phosphatases, and proteases. The differential binding of the enzymatic substrates and products to cationic polymers such as polyaraginine can be conveniently measured by fluorescence polarization.
Finally, we have discovered that the process of hybridization of peptide nucleic acid probes (PNAs) to their target DNA molecules can be followed by measuring the fluorescence polarization of a fluorophore attached to the PNA probes. These measurements can be done either in the presence or absence of polylysine in solution. Examples of the application of this method for single nucleotide polymorphism (SNP) typing are presented.
Diseases and changes in the way of life change the concentration and composition of the expired air. Our adaptable gas analyzer is intended for the selective analysis of expired air and can be adapted for the solution of current diagnostic and analytical tasks by the user (a physician or a patient). Having analyzed the existing trends in the development of noninvasive diagnostics we have chosen the method of noninvasive acetone detection in expired air, where the acetone concentration correlates with blood and urine glucose concentrations. The appearance of acetone in expired air is indicative of disorders that may be caused not only by diabetes but also be wrong diet, incorrect sportsmen training etc. To control the disorders one should know the acetone concentration in the human body. This knowledge allows one to judge upon the state of the patient, choose a correct diet that will not cause damage to the patient's health, determine sportsmen training efficiency and results and solve the artificial pancreas problem. Our device provide highly accurate analysis, rapid diagnostics and authentic acetone quantification in the patient's body at any time aimed at prediction of the patient's state and assessing the efficiency of the therapy used. Clinical implementation of the device will improve the health and save lives of many thousands of diabetes sufferers.