Browsing by Author "Lillehoj, Peter B."

Now showing 1 - 7 of 7
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    Electrochemical Biosensors for Cytokine Profiling: Recent Advancements and Possibilities in the Near Future
    (MDPI, 2021) Dutta, Nirmita; Lillehoj, Peter B.; Estrela, Pedro; Dutta, Gorachand
    Cytokines are soluble proteins secreted by immune cells that act as molecular messengers relaying instructions and mediating various functions performed by the cellular counterparts of the immune system, by means of a synchronized cascade of signaling pathways. Aberrant expression of cytokines can be indicative of anomalous behavior of the immunoregulatory system, as seen in various illnesses and conditions, such as cancer, autoimmunity, neurodegeneration and other physiological disorders. Cancer and autoimmune diseases are particularly adept at developing mechanisms to escape and modulate the immune system checkpoints, reflected by an altered cytokine profile. Cytokine profiling can provide valuable information for diagnosing such diseases and monitoring their progression, as well as assessing the efficacy of immunotherapeutic regiments. Toward this goal, there has been immense interest in the development of ultrasensitive quantitative detection techniques for cytokines, which involves technologies from various scientific disciplines, such as immunology, electrochemistry, photometry, nanotechnology and electronics. This review focusses on one aspect of this collective effort: electrochemical biosensors. Among the various types of biosensors available, electrochemical biosensors are one of the most reliable, user-friendly, easy to manufacture, cost-effective and versatile technologies that can yield results within a short period of time, making it extremely promising for routine clinical testing.
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    Finger-powered Microfluidic Mixer for Enhanced Electrochemical Detection of Protein Biomarkers
    (2024-04-11) Utzinger, Benjamin; Lillehoj, Peter B.
    This thesis presents the development of a finger-actuated micromixer to enhance the performance of an electrochemical immunosensor. Numerical simulations were performed to optimize the design of the mixer and study the flow characteristics upon actuation of the mixer. Using the optimized mixer design, a microfluidic finger-actuated mixer was fabricated and experiments were performed to visualize the flows generated by the mixer. The mixer was integrated with an electrochemical immunosensor for measurements of chemokine (C-X-C motif) ligand 9, a protein biomarker for autoimmune and inflammatory diseases, which was used for proof of principle. Experiments were performed to optimize the mixing and immunosensor parameters and to investigate the influence of the mixer on the immunosensor performance. Our results revealed that implementing mixing during the protein incubation steps accelerated biomolecular transport and enhanced antibody-antigen reactions, ultimately increasing the signal-to-background ratio and reducing the detection time. The ability to rapidly detect protein biomarkers with high sensitivity in a point‐of‐care format makes this device a promising tool for diagnostic testing.
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    Lateral flow immunochromatographic assay on a single piece of paper
    (Royal Society of Chemistry, 2021) Jiang, Xue; Lillehoj, Peter B.
    Lateral flow immunochromatographic assays (LFIAs) are analytical devices used to detect the presence of one or more target analytes in a liquid sample. While LFIAs are one of the simplest and inexpensive types of immunoassays, they consist of multiple components (sample pad, conjugate pad, membrane, absorbent pad, backing card) and materials, requiring time-consuming device assembly. Here, we report a unique lateral flow immunochromatographic assay constructed from a single piece of cellulose paper, which is fabricated via laser cutting. Compared with conventional lateral flow immunochromatographic devices, this single-layer immunoassay enables simpler and faster fabrication, while minimizing material consumption and overall device costs. For proof-of-concept, this device was used to detect Plasmodium falciparum histidine-rich protein 2 (PfHRP2), a biomarker for malaria infection, which could be detected at concentrations as low as 4 ng mL−1 by the naked eye with no cross reactivity with other common Plasmodium protein biomarkers. While offering similar speed and ease-of-use as conventional LFIAs with a higher detection sensitivity than existing LFIAs for PfHRP2 detection, this single-layer lateral flow immunoassay has the potential to improve malaria testing, as well as the detection of other important protein biomarkers for point-of-care testing.
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    Magnetic Nanoparticle-based Immunoassays for Rapid, High-Sensitivity Detection of Protein Biomarkers
    (2024-07-29) Singampalli, Kavya Lahari; Lillehoj, Peter B.; Grande-Allen, Kathryn J; Yee, Cassian
    The detection of protein biomarkers in human biofluids is a standard clinical practice for diagnosis, and monitoring of disease progression and therapeutic response. Current gold standard techniques for protein measurement are based on immunoassays, in which antibodies specific to the analyte of interest are used to bind and isolate the target protein. The most commonly used immunoassay is the enzyme-linked immunosorbent assay (ELISA), which produces a color change proportional to the concentration of the target analyte. A point-of-care friendly alternative to ELISA is an electrochemical amperometric immunosensor, which measures a current change. These techniques are highly sensitive, often detecting target analyte levels in the pg’s/mL, and specific due to their use of antibodies. However, they require long (3-4 hr) incubation times, skilled laboratory personnel, single-use substrates, and may not achieve the sensitivity necessary to detect ultralow levels of protein biomarkers. To address these challenges, we have employed magnetic nanoparticles to amplify the detection signal produced by immunoassays and enhance binding kinetics in both ELISA and electrochemical sensing. We have developed a magneto-ELISA employing dually-labelled magnetic nanoparticles (DMPs), which are bound to an excess of detection antibody (dAb) and enzymatic reporter, in order to increase the binding of target analytes, enhance signal amplification, and reduce incubation times within the assay. By using an external magnetic field, DMP-immunocomplexes are concentrated at the bottom of each well, facilitating binding with the capture antibody (cAb). Using Plasmodium falciparum histidine-rich protein 2 (PfHRP2), a marker for malaria, as a proof-of-concept biomarker, we found that this assay can detect proteins in the 10’s pg/mL range within 30 minutes, maintaining the sensitivity of a standard ELISA and producing results up to 4-fold faster. The magneto-ELISA was further adapted for serological detection of antibodies against Trypanosoma cruzi (T. cruzi), as would be found in chronic Chagas disease. Using DMPs conjugated to both Tc24, a protein specific to T. cruzi, and an enzymatic reporter, we show that anti-Tc24 antibodies can be identified in 6400x diluted clinical serum samples with an equivalent accuracy to a standard ELISA. We have also leveraged the capability of magnetic nanoparticles to be positioned using an external magnetic field to develop a reusable electrochemical sensor. Standard electrochemical immunoassay techniques render the sensors to be single-use consumables, increasing waste and costs associated with point-of-care quantitative protein biomarker detection. By using cAb-labelled magnetic nanoparticles (MNPs), we are able to temporarily immobilize immunocomplexes onto the working electrode, which can subsequently be washed away using a mild detergent. Furthermore, the addition of dually-labelled gold nanoparticles, bound to both dAb and a reporter molecule, allows for signal amplification. Together, these modifications to standard electrochemical sensing allow the sensor to be reused up to 100 times with a minimal reduction in analytical performance, while allowing for the detection of pg’s/mL of protein biomarkers in approximately one hour using point-of-care friendly instrumentation. Overall, we demonstrate the benefits of dually-labelled nanoparticles and MNPs in enhancing the sensitivity and reducing incubation times required for protein biomarker detection. The techniques described here are done with proof-of-concept biomarkers and biofluids, but can be easily adapted for the detection of other biomarkers. Furthermore, these techniques do not require additional laboratory equipment for protein detection, thus facilitating their adoption into clinical practice.
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    Microfluidic Magneto-Electrochemical Immunosensors for Rapid, High Sensitivity Quantification of Protein Biomarkers
    (2022-04-06) Li, Jiran; Lillehoj, Peter B.
    Electrochemical immunosensors are simple, portable diagnostic tools that are used for the detection and quantification of protein biomarkers. While electrochemical immunosensors are capable of rapid and high sensitivity measurements, they require complicated and/or long sample processing procedures or suffer from poor sensitivity using whole blood samples. Therefore, the goal of this research is to investigate new strategies to achieve rapid, high sensitivity protein measurements in complex biological matrices, such as blood serum and whole blood. This thesis introduces two novel electrochemical immunosensing platforms. The first is a microfluidic magneto-immunosensor based on a unique sensing scheme utilizing dual-labeled magnetic nanobeads for immunomagnetic enrichment and signal amplification. The functionality of this immunosensor was validated by using it to detect SARS-CoV-2 nucleocapsid protein, which could be detected at concentrations as low as 50 pg/ml in undiluted human serum in 55 minutes. This magneto-immunosensor was further developed into an electricity-free, mobile phone-based point-of-care (mPOC) diagnostic platform for the quantification of Plasmodium falciparum histidine-rich protein 2 (PfHRP2), a malaria parasite biomarker, which could be detected in whole blood at concentrations as low as 180 pg/mL in 15 minutes. The functionality of this mPOC platform was validated using 31 clinical blood samples obtained from malaria patients, which revealed its ability to quantify PfHRP2 with similar accuracy as a commercial ultrasensitive PfHRP2 ELISA kit. The second platform that was developed was an alternating current electrothermal flow (ACEF)- enhanced immunosensor, which utilizes electrothermal flow-induced mixing for enhanced immunocomplex formation and electrochemical detection. Proof of concept was carried out by performing measurements of buffer samples spiked with PfHRP2, which could be detected at concentrations as low as 1 ng/mL in 10 min. This ACEF-enhanced immunosensing technique was combined with our magneto-electrochemical immunosensor, which resulted in a significant improvement in the analytical performance, resulting in a lower limit of detection of 5.7 pg/mL in whole blood. Furthermore, each measurement could be completed in 7 minutes. In conclusion, we have presented several unique microfluidic magneto-electrochemical immunosensors capable of rapid, high sensitivity measurements in whole blood and blood-derived samples, which are promising diagnostic tools for rapid diagnostic testing and point-of-care testing.
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    Microneedle-based sampling of dermal interstitial fluid using a vacuum-assisted skin patch
    (Elsevier, 2024) Jiang, Xue; Wilkirson, Elizabeth C.; Bailey, Aaron O.; Russell, William K.; Lillehoj, Peter B.
    Interstitial fluid (ISF) contains a wealth of biomolecules, yet it is underutilized for diagnostic testing due to a lack of rapid and simple techniques for collecting abundant amounts of fluid. Here, we report a simple and minimally invasive technique for rapidly sampling larger quantities of ISF from human skin. A microneedle array is used to generate micropores in skin from which ISF is extracted using a vacuum-assisted skin patch. Using this technique, an average of 20.8 μL of dermal ISF is collected in 25 min, which is an ∼6-fold improvement over existing sampling methods. Proteomic analysis of collected ISF reveals that it has nearly identical protein composition as blood, and >600 medically relevant biomarkers are identified. Toward this end, we demonstrate the detection of SARS-CoV-2 neutralizing antibodies in ISF collected from COVID-19 vaccinees using two commercial immunoassays, showcasing the utility of this technique for diagnostic testing.
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    Microneedle-based skin patch for blood-free rapid diagnostic testing
    (Springer Nature, 2020) Jiang, Xue; Lillehoj, Peter B.
    Rapid diagnostic tests are one of the most commonly used tests to detect and screen for infectious diseases in the developing world. While these tests are simple, inexpensive, and readily available, they rely on finger-prick blood sampling, which requires trained medical personnel, poses risks of infection, and can complicate cooperation in young children, asymptomatic individuals, and communities with blood taboos. Here, we report a novel microneedle-based skin patch for the rapid detection of protein biomarkers in dermal interstitial fluid. Sample collection is facilitated by a hydrophilic hollow microneedle array that autonomously extracts and transports interstitial fluid to an antibody-based lateral flow test strip via surface tension for colorimetric antigen detection. We employ a simple gold enhancement treatment to enhance the detection sensitivity of this colloidal gold-based lateral flow assay and elucidate the underlying mechanism of this enhancement mechanism through experimental investigation. For proof-of-concept, this device was used to detect Plasmodium falciparum histidine-rich protein 2, a biomarker for malaria infection, which could be detected at concentrations as low as 8 ng/mL. Each test can be completed in <20 min and requires no equipment. To the best of our knowledge, this work is the first demonstration of a microneedle-based lateral flow assay for rapid protein detection in dermal interstitial fluid. In addition to its simplicity, minimally invasive nature, and low cost, this diagnostic device can be readily adapted to detect other protein biomarkers in interstitial fluid, making it a promising tool for point-of-care testing.