Sub-microliter Volumes Research Articles

Specific volume changes upon thermal phase transition of sub-microliter volumes of lipids and membrane proteins by a QCM nanopore sensor

Specific volume changes upon thermal phase transition of sub-microliter volumes of lipids and membrane proteins by a QCM nanopore sensor

Mapping the Potential of Microfluidics in Early Diagnosis and Personalized Treatment of Head and Neck Cancers.

Head and neck cancers (HNCs) account for ~4% of all cancers in North America and encompass cancers affecting the oral cavity, pharynx, larynx, sinuses, nasal cavity, and salivary glands. The anatomical complexity of the head and neck region, characterized by highly perfused and innervated structures, presents challenges in the early diagnosis and treatment of these cancers. The utilization of sub-microliter volumes and the unique phenomenon associated with microscale fluid dynamics have facilitated the development of microfluidic platforms for studying complex biological systems. The advent of on-chip microfluidics has significantly impacted the diagnosis and treatment strategies of HNC. Sensor-based microfluidics and point-of-care devices have improved the detection and monitoring of cancer biomarkers using biological specimens like saliva, urine, blood, and serum. Additionally, tumor-on-a-chip platforms have allowed the creation of patient-specific cancer models on a chip, enabling the development of personalized treatments through high-throughput screening of drugs. In this review, we first focus on how microfluidics enable the development of an enhanced, functional drug screening process for targeted treatment in HNCs. We then discuss current advances in microfluidic platforms for biomarker sensing and early detection, followed by on-chip modeling of HNC to evaluate treatment response. Finally, we address the practical challenges that hinder the clinical translation of these microfluidic advances.

In situ tumor cells detection using nanotube-functionalized & microfluidic-controlling multiresonance optical fiber

In situ and highly sensitive monitoring of tumor cells provides a fundamental understanding of the valuation of cancer diagnosis. However, existing techniques are largely unable to rapidly capture and precisely quantify the tumor cells. To address this, we propose a novel approach based on a nanotube-functionalized & microfluidic-controlling multiresonance optical fiber sensor. The sensor consists of a tilted fiber Bragg grating (TFBG) inscribed in the fiber core and specially designed halloysite nanotubes (HNTs) coated over the fiber surface. In particular, the HNTs are arranged in an orderly manner along the fiber surface to form slit-like patterned layers for enhanced the interaction with tumor cells, resulting in more effective capture of tumor cells. Such a sensor provides a powerful light scattering sensing ability. Based on the spectral area interrogation method, normal cells and tumor cells can be unambiguously discriminated within a few minutes, providing high sensitivity (limit of detection of 10 cells/mL), and a linear response for a large cell concentration range (10 ∼ 105 cells/mL). Meanwhile, by integrating the fiber sensor with a well-designed microfluidic chip, rapid and precise measurement of different tumor cell samples with low consumption (sub-microliter volumes) can be achieved, which provides a potential tool for cancer diagnosis and treatment.

Nano-liter perfusion microfluidic device made entirely by two-photon polymerization for dynamic cell culture with easy cell recovery

Polydimethylsiloxane (PDMS) has been the material of choice for microfluidic applications in cell biology for many years, with recent advances encompassing nano-scaffolds and surface modifications to enhance cell-surface interactions at nano-scale. However, PDMS has not previously been amenable to applications which require complex geometries in three dimensions for cell culture device fabrication in the absence of additional components. Further, PDMS microfluidic devices have limited capacity for cell retrieval following culture without severely compromising cell health. This study presents a designed and entirely 3D-printed microfluidic chip (8.8 mm × 8.2 mm × 3.6 mm) using two-photon polymerization (2PP). The ‘nest’ chip is composed of ten channels that deliver sub-microliter volume flowrates (to ~ 600 nL/min per channel) to 10 individual retrievable cell sample ‘cradles’ that interlock with the nest to create the microfluidic device. Computational fluid dynamics modelling predicted medium flow in the device, which was accurately validated by real-time microbead tracking. Functional capability of the device was assessed, and demonstrated the capability to deliver culture medium, dyes, and biological molecules to support cell growth, staining and cell phenotype changes, respectively. Therefore, 2PP 3D-printing provides the precision needed for nanoliter fluidic devices constructed from multiple interlocking parts for cell culture application.

Tissue harvest with a laser microbiopsy.

Traditional pathology workflow suffers from limitations including biopsy invasiveness, small fraction of large tissue samples being analyzed, and complex and time-consuming processing. We address limitations of conventional pathology workflow through development of a laser microbiopsy device for minimally invasive harvest of sub-microliter tissue volumes. Laser microbiopsy combined with rapid diagnostic methods, such as virtual hematoxylin and eosin (H&E) imaging has potential to provide rapid minimally invasive tissue diagnosis. Laser microbiopsies were harvested using an annular shaped Ho:YAG laser beam focused onto the tissue surface. As the annulus was ablated, the tissue section in the center of the annulus was ejected and collected directly onto a glass slide for analysis. Cryogen spray cooling was used before and after laser harvest to limit thermal damage. Microbiopsies were collected from porcine skin and kidney. Harvested microbiopsies were imaged with confocal microscopy and digitally false colored to provide virtual H&E images. Microbiopsies were successfully harvested from porcine skin and kidney. Computational and experimental results show the benefit of cryogen pre- and post-cooling to limit thermal damage. Virtual H&E images of microbiopsies retained observable cellular features including cell nuclei. Laser microbiopsy with virtual H&E imaging shows promise as a potential rapid and minimally invasive tool for biopsy and diagnosis.

Label-free monitoring of proteins in optofluidic hollow-core photonic crystal fibres

The fluorescent detection of proteins without labels or stains, which affect their behaviour and require additional genetic or chemical preparation, has broad applications to biological research. However, standard approaches require large sample volumes or analyse only a small fraction of the sample. Here we use optofluidic hollow-core photonic crystal fibres to detect and quantify sub-microlitre volumes of unmodified bovine serum albumin (BSA) protein down to 100 nM concentrations. The optofluidic fibre’s waveguiding properties are optimised for guidance at the (auto)fluorescence emission wavelength, enabling fluorescence collection from a 10 cm long excitation region, increasing sensitivity. The observed spectra agree with spectra taken from a conventional cuvette-based fluorimeter, corrected for the guidance properties of the fibre. The BSA fluorescence depended linearly on BSA concentration, while only a small hysteresis effect was observed, suggesting limited biofouling of the fibre sensor. Finally, we briefly discuss how this method could be used to study aggregation kinetics. With small sample volumes, the ability to use unlabelled proteins, and continuous flow, the method will be of interest to a broad range of protein-related research.

Single bubble in-tube microextraction coupled with capillary electrophoresis.

Headspace (HS) extraction is a sample pretreatment technique for volatile and semivolatile organic compounds in a complex matrix. Recently, in-tube microextraction (ITME) coupled with CE using an acceptor plug placed in the capillary inlet was developed as a simple but powerful HS extraction method. Here, we present single bubble (SB) ITME using a bubble hanging to the capillary inlet immersed in a sample donor solution as a HS of submicroliter volume (∼200 nL). The analytes evaporated to the bubble were extracted into the acceptor phase through the capillary opening, then electrophoresis of the enriched extract was carried out. Since the bubble volume was much smaller than a conventional HS volume (∼1mL), it was filled with the evaporated analytes rapidly and the analytes could be enriched much faster compared to conventional HS-ITME. Owing to the high surface-to-volume ratio of the SB, 5min SB-ITME yielded the enrichment factor values similar to those of 10min HS-ITME. When 5min SB-ITME at room temperature was applied to a tap water sample, the enrichment factors of 2,4,6-trichlorophenol (TCP), 2,3,6-TCP, and 2,6-dichlorophenol were 53, 41, and 60, respectively, and the LOQs obtained by monitoring the absorbance at 214nm were 5.6-8.3 ppb, much lower than 200 ppb, the World Health Organization guideline for the maximum permissible concentration of 2,4,6-TCP in drinking water.

Microfluidic Systems for Cancer Diagnosis and Applications.

Microfluidic devices have led to novel biological advances through the improvement of micro systems that can mimic and measure. Microsystems easily handle sub-microliter volumes, obviously with guidance presumably through laminated fluid flows. Microfluidic systems have production methods that do not need expert engineering, away from a centralized laboratory, and can implement basic and point of care analysis, and this has attracted attention to their widespread dissemination and adaptation to specific biological issues. The general use of microfluidic tools in clinical settings can be seen in pregnancy tests and diabetic control, but recently microfluidic platforms have become a key novel technology for cancer diagnostics. Cancer is a heterogeneous group of diseases that needs a multimodal paradigm to diagnose, manage, and treat. Using advanced technologies can enable this, providing better diagnosis and treatment for cancer patients. Microfluidic tools have evolved as a promising tool in the field of cancer such as detection of a single cancer cell, liquid biopsy, drug screening modeling angiogenesis, and metastasis detection. This review summarizes the need for the low-abundant blood and serum cancer diagnosis with microfluidic tools and the progress that has been followed to develop integrated microfluidic platforms for this application in the last few years.

Aqueous two-phase deposition and fibrinolysis of fibroblast-laden fibrin micro-scaffolds

This paper describes printing of microscale fibroblast-laden matrices using an aqueous two-phase approach that controls thrombin-mediated enzymatic crosslinking of fibrin. Optimization of aqueous two-phase formulations enabled polymerization of consistent sub-microliter volumes of cell-laden fibrin. When plasminogen was added to these micro-scaffolds, the primary normal human lung fibroblasts converted it to plasmin, triggering gradual degradation of the fibrin. Time-lapse live-cell imaging and automated image analysis provided readouts of time to degradation of 50% of the scaffold as well as maximum degradation rate. The time required for degradation decreased linearly with cell number while it increased in a dose-dependent manner upon addition of TGF-β1. Fibroblasts isolated from idiopathic pulmonary fibrosis patients showed similar trends with regards to response to TGF-β1 stimulation. Addition of reactive oxygen species (ROS) slowed fibrinolysis but only in the absence of TGF-β1, consistent with published studies demonstrating that pro-fibrotic cellular phenotypes induced by TGF-β1 are mediated, at least in part, through increased production of ROS. FDA-approved and experimental anti-fibrosis drugs were also tested for their effects on fibrinolysis rates. Given the central role of fibrinolysis in both normal and pathogenic wound healing of various tissues, the high-throughput cell-mediated fibrinolysis assay described has broad applicability in the study of many different cell types and diseases. Furthermore, aqueous two-phase printing of fibrin addresses several current limitations of fibrin bio-inks, potentially enabling future applications in tissue engineering and in vitro models.

Catecholamine-Copper Redox as a Basis for Site-Specific Single-Step Functionalization of Material Surfaces.

Realization of robust and facile surface functionalization processes is critical to biomaterials and biotechnology yet remains a challenge. Here, we report a new chemical approach that enables operationally simple and site-specific surface functionalization. The mechanism involves a catechol-copper redox chemistry, where the oxidative polymerization of an alkynyl catecholamine reduces Cu(II) to Cu(I), which in situ catalyzes a click reaction with azide-containing molecules of interest (MOIs). This process enables drop-coating and grafting of two- and three-dimensional solid surfaces in a single operation using as small as sub-microliter volumes. Generalizability of the method is shown for immobilizing MOIs of diverse structure and chemical or biological activity. Biological applications in anti-biofouling, cellular adhesion, scaffold seeding, and tissue regeneration are demonstrated, in which the activities or fates of cells are site-specifically manipulated. This work advances surface chemistry by integrating simplicity and precision with multipurpose surface functionalization.

Autonomous, Real-Time Monitoring Electrochemical Aptasensor for Circadian Tracking of Cortisol Hormone in Sub-microliter Volumes of Passively Eluted Human Sweat.

The proposed work involves the development of an autonomous, label-free electrochemical sensor for real-time monitoring of cortisol levels expressed naturally in sub-microliter sweat volumes, for prolonged sensing periods of ∼8 h. Highly specific single-stranded DNA (ssDNA) aptamer is used for affinity capture of cortisol hormone eluted in sweat dynamically. The cortisol present in sweat binds to the aptamer capture probe that changes conformation and modulates electrochemical properties at the electrode-buffer interface, which was studied using dynamic light scattering studies for the entire physiological sweat pH. Attenuated total reflectance-Fourier transform infrared spectroscopy and UV-vis spectroscopy were used to optimize the binding chemistry of the elements of the sensor stack. Nonfaradaic electrochemical impedance spectroscopy was used to calibrate the sensor for a dynamic range of 1-256 ng/mL. An R2 of 0.97 with an output signal range of 20-50% was obtained. Dynamic cortisol level variation tracking was studied using continuous dosing experiments to calibrate the sensor for temporal variation. The sensor did not show significant susceptibility to noise due to cross-reactive interferents and nonspecific buffer constituents. The performance of the developed aptasensor was compared with the previously established cortisol immunosensor in terms of surface charge behavior and nonfaradaic biosensing. The aptamer sensor shows a higher signal-to-noise ratio, better resolution, and has a larger output range for the same input range as the cortisol immunosensor. The feasibility of deploying the developed aptasensing scheme as continuous lifestyle and performance monitors was validated through human subject studies.

An isotope ratio mass spectrometry-based method for hydrogen isotopic analysis in sub-microliter volumes of water: Application for multi-isotope investigations of gases extracted from fluid inclusions.

The study of multi-isotope systematics of fluid inclusions is of great importance for understanding of the sources and evolution of fluid phases in mantle rocks and ore deposits. The most appropriate technique for such investigations is a (stepwise) crushing method that is widely used for noble gases and nitrogen. However, because of the possible influence of mechanochemical reactions and back sorption, analyses of the isotope composition of water extracted by crushing from fluid inclusions are challenging. An isotope ratio mass spectrometry (IRMS)-based method for hydrogen (and oxygen) isotopic analysis in sub-microliter volumes of water extracted from fluid inclusions by crushing is presented. The verification of the possible influence of adsorption processes and mechanochemical reactions on the results of isotope analysis was performed for the first time. For that a series of parallel analyses of hydrogen isotopic ratios from water inclusions in quartz applying physically different extraction methods (crushing and thermodecrepitation) was conducted. Four series of quartz aliquots were analyzed: three series extracting water by crushing (two series for δ2 H values and one for δ18 O values) and one by thermodecrepitation. The mean value for the crushing results is δ2 H = -85.3 ± 3.6 ‰ (1σ, n = 11), which coincides well with the thermodecrepitation data (-86.3 ± 2.0 ‰, 1σ, n = 5), suggesting that our methodological approach allows the influence of back sorption or mechanochemical reactions during the crushing experiment to be minimized. The reproducibility of the oxygen isotopic ratios is ±0.9 ‰ (1σ, n = 5). The conducted experiments have shown that the influence of back sorption or mechanochemical reactions during crushing on isotopic results is not crucial for our method. The developed IRMS-based method for hydrogen (and oxygen) isotopic analysis in sub-microliter volumes of water is well applicable for multi-isotope investigations of gases extracted from fluid inclusions. As an application a well-defined 40 Ar/36 Ar-δ2 H correlation in mantle rocks is presented for the first time.

Dialysis of one sample drop on-line connected with electrophoresis in short capillary

An analytical apparatus is described, based on on-line connection of electrophoresis in a short capillary with a dialysis unit enabling dialysis in micro-litre sample volumes into submicro-litre volumes of an acceptor solution in a dialysing fibre. After a defined dialysis time, the dialysate from the dialysing fibre is injected into a separation capillary through an air-assisted flow-gating interface cast from PDMS. In the flow-gating injection space, the exit from the delivery capillary bringing the dialysate is placed directly opposite the entrance into the separation capillary at a distance of 380 μm. In order to enable injection of a very small volume of dialysate, the background electrolyte is forced out of the injection space with air before the injection, so that a drop of dialysate with a volume of about 0.1 μL is formed between the exit from the delivery and the entrance into the separation capillary; the dialysate is injected hydrodynamically from this dialysate drop. Then the injection space is filled with the background electrolyte and the separation is commenced. The basic properties of the apparatus were tested on model mixtures of inorganic cations (K+, Ba2+ and Na+) and organic molecules (creatinine, histidine and arginine). The applicability to real samples was tested on the determination of basic amino acids (histidine, lysine and arginine) in a blood serum sample.

Design and Comparison of Resonant and Non-Resonant Single-Layer Microwave Heaters for Continuous Flow Microfluidics in Silicon-Glass Technology

This paper presents a novel concept for the co-design of microwave heaters and microfluidic channels for sub-microliter volumes in continuous flow microfluidics. Based on the novel co-design concept, two types of heaters are presented, co-designed and manufactured in high-resistivity silicon-glass technology, resulting in a building block for consumable and mass-producible micro total analysis systems. Resonant and non-resonant co-planar waveguide transmission line heaters are investigated for heating of sub-micro-liter liquid volumes in a channel section at 25 GHz. The heating rates of 16 and 24 °C/s are obtained with power levels of 32 dBm for the through line and the open-ended line microwave heater, respectively. The heating uniformity of developed devices is evaluated with a Rhodamine B and deionized water mixture on a micrometer scale using the microwave-optical measurement setup. Measurement results showed a good agreement with simulations and demonstrated the potential of microwave heating for microfluidics.

Effect of reducing sample volume on the detection of drugs in urine by transmission mode direct analysis in real time mass spectrometry.

Matrix interference attributed to urea and other nitrogenous substances in unprocessed urine is significant. In this study desorption ionization of sub-microliter volume samples is performed in an effort to improve the detection of drugs in unprocessed urine using transmission mode-direct analysis in real time mass spectrometry (TM-DART-MS). Urine samples were spiked with analytical standards of two drugs of abuse, codeine and methadone. Various sub-microliter volumes of unprocessed urine were deposited onto wire mesh screen consumables and analyzed using TM-DART for desorption ionization and a high-resolution mass spectrometer operated in full scan mode for mass analysis. A 22 factorial design of experiment (DOE) was employed to examine the effects of sample volume and sample introduction speed to the DART source. Results from analysis of one microliter and sub-microliter sample volumes were compared by measuring the signal produced by TM-DART-MS. Based on an α of 0.05, the lower-volume samples yielded spectra where the abundance of urea and creatinine ions was reduced, thus significantly improving the TM-DART-MS signal for drugs of abuse. Using slower sample introduction speeds increased the time during which the sample was exposed to the heated ionization gas, resulting in a significant increase in the TM-DART-MS signal. Reducing the sample volume to sub-microliter levels improved the detection of drugs of abuse present as either individual or multiple components of the untreated urine. The improved signal demonstrates the potential for using sub-microliter volumes for screening drugs in urine without the need for chromatography or sample pretreatment.

A Novel in vitro Model for Prostate Cancer Bone Metastasis using Microfluidics

Alexander Cui, Srinivasa Rao, Sam Olechnowicz, James Edwards, Peter Cook, Edmond Walsh, Claire Edwards
 Prostate cancer (PCa) cells predominantly metastasise to bone, and once this happens the disease is incurable. Hence it is vital to understand the mechanism of bone metastasis in PCa. Several models have been established to study PCa bone metastasis in vitro, involving the co-culture of PCa cell lines with cells of the bone microenvironment such as osteoblasts, osteoclasts, bone marrow stromal cells, etc. However, these models are limited to one-to-one co-culture (i.e., PCa cells co-cultured with one other cell type), which overly simplifies the complex bone microenvironment consisting of multiple cell types.
 We aimed to overcome this limitation by adapting a microfluidics platform that employs a novel mechanism to isolate sub-microliter volumes using fluid walls. By using this technology, we were able to successfully differentiate osteoblasts and adipocytes from precursor cells (2T3 and ST2 cells respectively) in nanowells, and co-cultured them with PC3 prostate cancer cells. Differentiation to osteoblasts and adipocytes was confirmed by calcein green and BODIPY live staining which could be monitored using the Incucyte imaging platform.
 Following co-culture, the PCa cells were trypsinised and total RNA was extracted from them. A cDNA amplification step was successfully incorporated into the reverse transcription protocol to provide sufficient material for quantitative PCR analysis. Further, sequencing adapters were added to the cDNA to generate an RNA-sequencing library for transcriptome-wide analysis. Using this novel model, with imaging, RNA-sequencing and qPCR as read-outs, we are working on elucidating the signalling pathways that are activated by the bone microenvironment in PCa cells.

Counting-based microfluidic paper-based devices capable of analyzing submicroliter sample volumes.

In this paper, we report the development of semiquantitative counting-based lateral flow assay (LFA)-type microfluidic paper-based analytical devices ( PADs) to analyze samples at submicroliter volumes. The ability to use submicroliter sample volumes is a significant advantage for PADs since it enables enhanced multiplexing, reduces cost, and increases user-friendliness since small sample volumes can be collected using methods that do not require trained personnel, such as finger pricking and microneedles. The challenge of accomplishing a semiquantitative test readout using submicroliter sample volumes was overcome with a counting-based approach. In order to use submicroliter sample volumes, we developed a flow strategy with a running liquid to facilitate flow through the assay. The efficacy of the devices was confirmed with glucose and total human immunoglobulin E (IgE) tests using 0.5 l and 1 l of sample solutions, respectively. Semiquantitative results were generated to predict glucose concentrations in the range of 0-12 mmol/l and IgE concentrations in the range of 0-400 ng/ml. The counting-based approach correlates the number of dots that exhibited a color change to the concentration of the analyte, which provides a more user-friendly method as compared with interpreting the intensity of a color change. The devices reported herein are the first counting-based LFA-type PADs capable of semiquantitative testing using submicroliter sample volumes.

Real-time laser induced chemical derivatizations of peptide N-Terminus for in-situ mass spectrometric sequencing at sub-picomole and nanosecond scale

Distinguishing b- and y-ions is essential to compute amino acid sequences from either N- or C-terminus in mass spectrometry. We described herein a solvent free and real time on-plate derivatization approach that can tag N-terminus of peptides at microliter level with p-chlorobenzaldehyde or 2-hydroxy-5-methylisophthalaldehyde for matrix assisted laser desorption ionization mass spectrometry (MALDI MS). Less than 1 μL of sample solutions can be directly mixed with equal volumes of p-chlorobenzaldehyde or 2-hydroxy-5-methylisophthalaldehyde and α-cyano-4-hydroxycinnamic acid (CHCA), a matrix compound to co-crystalize with analytes for efficient absorption of laser energy and peptide ionization. When the mixture spotted on the sample plate is irradiated with the 3rd harmonic (355 nm) of Nd3+:YAG laser pulses (3 ns width), N-terminal amine groups of peptides instantly react with carbonyl groups of chlorobenzaldehyde or 2-hydroxy-5-methylisophthalaldehyde. Resultant peptides carrying with on-plate formed azomethine group (-CN-) are simultaneously protonated and isolated as precursor ions for subsequent collision-activated dissociation. The mass shift with unique Cl isotopic signature unambiguously distinguishes b ions from y ions and other ions. This method does not need extensive sample preparation and is useful for those samples with limited quantities down to sub-picomole level in sub-microliter volumes. The efficiency was demonstrated with synthetic peptides and tryptic peptides of model proteins. It was found that 2-hydroxy-5-methylisophthalaldehyde provides improved yield for peptides containing lysine residues. Unknown proteins of human saliva and bovine milk as well as phosphopeptides have been identified.

Rapid Chemical Reaction Monitoring by Digital Microfluidics-NMR: Proof of Principle Towards an Automated Synthetic Discovery Platform.

Microcoil nuclear magnetic resonance (NMR) has been interfaced with digital microfluidics (DMF) and is applied to monitor organic reactions in organic solvents as a proof of concept. DMF permits droplets to be moved and mixed inside the NMR spectrometer to initiate reactions while using sub-microliter volumes of reagent, opening up the potential to follow the reactions of scarce or expensive reagents. By setting up the spectrometer shims on a reagent droplet, data acquisition can be started immediately upon droplet mixing and is only limited by the rate at which NMR data can be collected, allowing the monitoring of fast reactions. Here we report a cyclohexene carbonate hydrolysis in dimethylformamide and a Knoevenagel condensation in methanol/water. This is to our knowledge the first time rapid organic reactions in organic solvents have been monitored by high field DMF-NMR. The study represents a key first step towards larger DMF-NMR arrays that could in future serve as discovery platforms, where computer controlled DMF automates mixing/titration of chemical libraries and NMR is used to study the structures formed and kinetics in real time.

Hemolysis Detection in Sub-Microliter Volumes of Blood Plasma

Hemolysis is one of the main reasons for blood sample rejection in clinical laboratories. The scope of this study is to develop a simple, sensitive, and cost-effective method for hemolysis detection in presence of interferants like bilirubin, and lipids in sub-microliter volumes of blood plasma. Experimental samples were prepared in plasma extracted from whole blood. These samples were typically mixtures of hemoglobin, bilirubin, and lipids of varying concentrations. A multispectral optical setup was devised to probe these samples without any optical filters or moving parts. The hemoglobin content in the samples were measured within the range of 0mg/dL to 400 mg/dL concentration using <1μL of detection volume. Detection sensitivity of >90% accuracy and ∼10% coefficient of variation across 27 unknown samples. The optical attenuation path length used was merely ∼150 micrometers. The spectral interference due to overlapping absorption spectrum of bilirubin and scattering spectrum of lipids are resolved using linear matrix algebra algorithms. Hemolysis can lead to spurious measurements of key clinical parameters such as Potassium, Lactate Dehydrogenase, Aspartic Acid Transferase, and other diagnostic biomarkers. Commonly used visual inspection of blood plasma coloration is prone to variability. Description of the sample preparation, calibration and verification of the experimental setup and linear matrix algebra algorithm for analyte interference determination is reported here. Owing to the sub-microliter detection volume and high sensitivity, the system has realistic potential to be implemented in point of care medical devices that demands such low volumes of clinical specimen.