Degradation of hydrogel beads for the detection of serum bicarbonate levels for the diagnosis of metabolic alkalosis at the point of care.

In this work, we present a novel point-of-care hydrogel-based diagnostic device for the rapid detection of elevated bicarbonate levels in serum for the diagnosis of mild to severe cases of metabolic alkalosis. Our system consists of hydrogel beads composed of calcium alginate and the nonionic polymer dextran. This assay utilizes the reaction of sodium bicarbonate and citric acid to produce citrate, a metal chelator capable of competitively binding to calcium cations in the gel matrix to trigger hydrogel degradation. This results in successful detection of elevated bicarbonate concentrations in less than one hour. Specifically, critically high bicarbonate concentrations of 50, 45, and 40 mmol L-1 in human serum were detected in as little as 10, 15, and 20 min, respectively. To demonstrate the assay's feasibility for use in resource-limited settings, we developed a simple electronic device that achieved similar results and could be used by untrained individuals with no lab equipment and minimal power. To our knowledge, this is the first demonstration of the use of nonionic polymers to synthesize and improve the morphology of calcium alginate hydrogel beads using a simple processing method that involves minimal labor and equipment. The simplified bead synthesis protocol combined with the user-friendly device allows for the rapid detection of metabolic alkalosis at the point of care.

Application of the aqueous two-phase system and nanozyme signal enhancement for the improved detection of Plasmodium lactate dehydrogenase in serum.

Malaria is an infectious disease that can cause severe sickness and death if not diagnosed and treated in a timely manner. The current gold standard technique for malaria diagnosis is microscopy, which requires a dedicated laboratory setting and trained personnel and can have a long time to result. These requirements can be alleviated using paper-based diagnostic devices that enable rapid and inexpensive diagnosis at the point of care, which can allow patients to receive treatment before their symptoms progress when used for early detection of diseases. The lateral-flow immunoassay (LFA) is one such device, but currently available LFAs are susceptible to false negative results caused by low parasite density. To improve sensitivity and detection, we utilized the aqueous two-phase system (ATPS) to concentrate and purify the sample, and nanozyme signal enhancement to increase the intensity of the visible signal on the test strip. We were able to achieve a limit of detection (LOD) of 0.01 ng/mL for the malaria biomarker Plasmodium lactate dehydrogenase (pLDH) in human serum using a multi-step assay combining the LFA format with the ATPS and nanozyme signal enhancement.

Combination of the lateral-flow immunoassay with multicolor gold nanorod etching for the semi-quantitative detection of digoxin.

We are the first to combine the lateral-flow immunoassay (LFA) with gold nanorod (GNR) etching to achieve a multicolor readout where the color produced was correlated with digoxin concentrations in human serum in the relevant range for therapeutic drug monitoring of 0.5-3.0 ng mL-1.

On-demand nanozyme signal enhancement at the push of a button for the improved detection of SARS-CoV-2 nucleocapsid protein in serum.

We developed an innovative 3D printed casing that incorporates a lateral-flow immunoassay, dehydrated signal enhancement reagents, and a sealed buffer chamber. With only the push of a button for signal enhancement, our device detected the SARS-CoV-2 N-protein in 40 min at concentrations as low as 0.1 ng mL-1 in undiluted serum.

Polymeric micelles using cholinium-based ionic liquids for the encapsulation and release of hydrophobic drug molecules.

We generated stable amphiphilic copolymer-based polymeric micelles (PMs) with temperature-responsive properties utilizing Pluronic® L35 and a variety of ionic liquids (ILs) to generate different aqueous two-phase micellar systems (ATPMSs). The partitioning of the hydrophobic model compound curcumin (CCM) into the PM-rich phase and the drug delivery capabilities of the PMs were investigated. ATPMSs formed using more hydrophobic ILs (i.e., [Ch][Hex] ≈ [Ch][But] > [Ch][Pro] > [Ch][Ac] ≈ [Ch]Cl) were the most effective in partitioning (KCCM) and recovering (RECRich) CCM into the PM-rich phase (15.2 < KCCM < 22.0 and 90% < RECRich < 95%, respectively). Moreover, using 1.2 M [Ch][But] and 0.2 M [Ch][Hex] ILs yielded higher encapsulation efficiency (EE) (94.1 and 96.0%, respectively) and drug loading (DL) capacity (14.8 and 16.2%, respectively), together with an increase in the average hydrodynamic diameter of the PMs (DH) (42.5 and 45.6 nm, respectively). The CCM-PM formulations were stable at 4.0, 25.0, and 37.0 °C and the release of CCM was faster with the less hydrophobic ILs (i.e., [Ch]Cl and [Ch][Ac]). Furthermore, due to the lower critical solution temperature properties of Pluronic® L35, the PMs exhibit temperature responsiveness at 37.0 °C. In vitro cytotoxicity assays were also performed to determine the potency of CCM-PM formulations, and a 1.8-fold decrease in IC50 values was observed between the CCM-PMs/[Ch][Hex] and CCM-PMs/[Ch]Cl formulations for PC3 cells. The lower IC50 value for the [Ch][Hex] version corresponded to a greater potency compared to the [Ch]Cl version, since a lower concentration of CCM was required to achieve the same therapeutic effect. The ATPMSs investigated in this study serve as a novel platform for Pluronic® L35/PBS buffer (pH 7.4) + IL-based ATPMS development. The unique properties reported here may be useful in applications such as controlled-release drug delivery systems (DDS), encapsulation, and bioseparations.

Point-of-Need Diagnostics for Foodborne Pathogen Screening.

Foodborne illness is a major public health issue that results in millions of global infections annually. The burden of such illness sits mostly with developing countries, as access to advanced laboratory equipment and skilled lab technicians, as well as consistent power sources, is limited and expensive. Current gold standards in foodborne pathogen screening involve labor-intensive sample enrichment steps, pathogen isolation and purification, and costly readout machinery. Overall, time to detection can take multiple days, excluding the time it takes to ship samples to off-site laboratories. Efforts have been made to simplify the workflow of such tests by integrating multiple steps of foodborne pathogen screening procedures into a singular device, as well as implementing more point-of-need readout methods. In this review, we explore recent advancements in developing point-of-need devices for foodborne pathogen screening. We discuss the detection of surface markers, nucleic acids, and metabolic products using both paper-based and microfluidic devices, focusing primarily on developments that have been made between 2015 and mid-2020.

Developments in integrating nucleic acid isothermal amplification and detection systems for point-of-care diagnostics.

Early disease detection through point-of-care (POC) testing is vital for quickly treating patients and preventing the spread of harmful pathogens. Disease diagnosis is generally accomplished using quantitative polymerase chain reaction (qPCR) to amplify nucleic acids in patient samples, permitting detection even at low target concentrations. However, qPCR requires expensive equipment, trained personnel, and significant time. These resources are not available in POC settings, driving researchers to instead utilize isothermal amplification, conducted at a single temperature, as an alternative. Common isothermal amplification methods include loop-mediated isothermal amplification, recombinase polymerase amplification, rolling circle amplification, nucleic acid sequence-based amplification, and helicase-dependent amplification. There has been a growing interest in combining such amplification methods with POC detection methods to enable the development of diagnostic tests that are well suited for resource-limited settings as well as developed countries performing mass screenings. Exciting developments have been made in the integration of these two research areas due to the significant impact that such approaches can have on healthcare. This review will primarily focus on advances made by North American research groups between 2015 and June 2020, and will emphasize integrated approaches that reduce user steps, reliance on expensive equipment, and the system's time-to-result.

Rapid Diagnostic Test Kit for Point-of-Care Cerebrospinal Fluid Leak Detection.

Cerebrospinal fluid (CSF) leaks can occur when there is communication between the intracranial cavities and the external environment. They are a common and serious complication of numerous procedures in otolaryngology, and if not treated, persistent leaks can increase a patient's risk of developing life-threatening complications such as meningitis. As it is not uncommon for patients to exhibit increased secretions postoperatively, distinguishing normal secretions from those containing CSF can be difficult. Currently, there are no proven, available tests that allow a medical provider concerned about a CSF leak to inexpensively, rapidly, and noninvasively rule out the presence of a leak. The gold standard laboratory-based test requires that a sample be sent to a tertiary site for analysis, where days to weeks may pass before results return. To address this, our group recently developed a semiquantitative, barcode-style lateral-flow immunoassay (LFA) for the quantification of the beta-trace protein, which has been reported to be an indicator of the presence of CSF leaks. In the work presented here, we created a rapid diagnostic test kit composed of our LFA, a collection swab, dilution buffers, disposable pipettes, and instructions. Validation studies demonstrated excellent predictive capabilities of this kit in distinguishing between clinical specimens containing CSF and those that did not. Our diagnostic kit for CSF leak detection can be operated by an untrained user, does not require any external equipment, and can be performed in approximately 20 min, making it well suited for use at the point of care. This kit has the potential to transform patient outcomes.

Automation of Biomarker Preconcentration, Capture, and Nanozyme Signal Enhancement on Paper-Based Devices.

Infectious diseases remain one of the leading causes of deaths in developing countries because of a lack of basic sanitation, healthcare clinics, and centralized laboratories. Paper-based rapid diagnostic tests, such as the lateral-flow immunoassay (LFA), provide a promising alternative to the traditional laboratory-based tests; however, they typically suffer from having a poor sensitivity. Biomarker preconcentration and signal enhancement are two common methods to improve the sensitivity of paper-based assays. While effective, these methods often require multiple liquid handling steps which are not ideal for use by untrained personnel in a point-of-care setting. Our lab previously discovered the phenomenon of an aqueous two-phase system (ATPS) separating on paper, which allowed for the seamless integration of concentration and detection of biomarkers on the LFA. In this work, we have extended the functionality of an ATPS separating on paper to automate the sequential delivery of signal enhancement reagents in addition to concentrating biomarkers. The timing of reagent delivery was controlled by changing the initial composition of the ATPS. We applied this technology to automate biomarker concentration and nanozyme signal enhancement on the LFA, resulting in a 30-fold improvement in detection limit over the conventional LFA when detecting Escherichia coli, all while maintaining a single application step.

Controlling Macroscopic Phase Separation of Aqueous Two-Phase Polymer Systems in Porous Media.

In previous work, our group discovered a phenomenon in which a mixed polymer-salt or mixed micellar aqueous two-phase system (ATPS) separates into its two constituent phases as it flows within paper. While these ATPSs worked well in their respective studies to concentrate the target biomarker and improve the sensitivity of the lateral-flow immunoassay, different ATPSs can be advantageous for new applications based on factors such as biomarker partitioning or biochemical compatibility between ATPS and sample components. However, since the mechanism of phase separation in porous media is not completely understood, introducing other ATPSs to paper is an unpredictable process that relies on trial and error experiments. This is especially true for polymer-polymer ATPSs in which the characteristics of the two phases appear quite similar. Therefore, our group aimed to develop semiquantitative guidelines for choosing ATPSs that can phase separate in paper. In this work, we evaluated the Washburn equation and its parameters as a potential mathematical framework to describe the flow behavior of polymer-salt and micellar ATPSs in fiberglass paper. We compared bulk phase fluid characteristics and identified the viscosity difference between the phases as a key determinant of the potential for phase separation in paper. We then used this parameter to predict the phase separation capabilities of polyethylene glycol (PEG)-dextran ATPSs in paper and control the composition of the leading and lagging phases. We also, for the first time, successfully demonstrated the phase separation phenomenon in hydrogels, thereby extending its application and potential benefits to an alternative porous medium.

Ionic Liquid Aqueous Two-Phase Systems for the Enhanced Paper-Based Detection of Transferrin and Escherichia coli.

Aqueous two-phase systems (ATPSs) have been widely utilized for liquid-liquid extraction and purification of biomolecules, with some studies also demonstrating their capacity as a biomarker concentration technique for use in diagnostic settings. As the limited polarity range of conventional polymer-based ATPSs can restrict their use, ionic liquid (IL)-based ATPSs have been recently proposed as a promising alternative to polymer-based ATPSs, since ILs are regarded as tunable solvents with excellent solvation capabilities for a variety of natural compounds and proteins. This study demonstrates the first application of IL ATPSs to point-of-care diagnostics. ATPSs consisting of 1-butyl-3-methylimidazolium tetrafluoroborate ([Bmim][BF4]) and sodium phosphate salt were utilized to quickly concentrate biomarkers prior to detection using the lateral-flow immunoassay (LFA). We found the phase separation speed of the IL ATPS to be very rapid and a significant improvement upon the separation speed of both polymer-salt and micellar ATPSs. This system was successfully applied to both sandwich and competitive LFA formats and enhanced the detection of both Escherichia coli bacteria and the transferrin protein up to 8- and 20-fold, respectively. This system's compatibility with a broad range of biomolecules, rapid phase separation speed, and tunability suggest wide applicability for a large range of different antigens and biomarkers.

Point-of-Care Cerebrospinal Fluid Detection.

OBJECTIVE: A cerebrospinal fluid leak is one of the most serious complications in otolaryngology. It may occur as a result of injury to the skull base, typically traumatic or iatrogenic. While the presence of a leak is often discerned in the emergent setting, distinguishing normal secretions from those containing cerebrospinal fluid can be difficult during postoperative visits in the clinic. As most current laboratory-based assays are labor intensive and require several days to result, we aim to develop a more user-friendly and rapid point-of-care cerebrospinal fluid detection device. STUDY DESIGN: Our laboratory developed a barcode-style lateral-flow immunoassay utilizing antibodies for beta-trace protein, a protein abundant in and specific for cerebrospinal fluid, with a concentration of 1.3 mg/L delineating a positive result. SETTING: Tertiary medical center. SUBJECTS AND METHODS: Tests with known concentrations of resuspended beta-trace protein and the contents of discarded lumbar drains (presumed to contain cerebrospinal fluid) were performed to validate our novel device. RESULTS: Our results demonstrate the ability of our device to semiquantitatively identify concentrations of beta-trace protein from 0.3-90 mg/L, which is within the required range to diagnose a leak, thus making beta-trace protein an excellent target for rapid clinical detection. CONCLUSION: Herein we detail the creation and initial validation of the first point-of-care cerebrospinal fluid detection device. This device is a feasible method to more efficiently and cost-effectively identify cerebrospinal fluid leaks, minimize costs, and improve patient outcomes.

A one-pot, isothermal DNA sample preparation and amplification platform utilizing aqueous two-phase systems.

Infectious diseases remain one of the major causes of death worldwide in developing countries. While screening via conventional polymerase chain reaction (PCR) is the gold standard in laboratory testing, its limited applications at the point-of-care have prompted the development of more portable nucleic acid detection systems. These include isothermal DNA amplification techniques, which are less equipment-intensive than PCR. Unfortunately, these techniques still require extensive sample preparation, limiting user accessibility. In this study, we introduce a novel system that combines thermophilic helicase-dependent amplification (tHDA) with a Triton X-100 micellar aqueous two-phase system (ATPS) to achieve cell lysis, lysate processing, and enhanced nucleic acid amplification in a simple, one-step process. The combined one-pot system was able to amplify and detect a target gene from whole-cell samples containing as low as 102 cfu/mL, and is the first known application of ATPSs to isothermal DNA amplification. This system's ease-of-use and sensitivity underlie its potential as a point-of-care diagnostic platform to detect for infectious diseases. Graphical abstract ᅟ.

A Combined Aqueous Two-Phase System and Spot-Test Platform for the Rapid Detection of Escherichia coli O157:H7 in Milk.

Foodborne illnesses are a public health concern in the United States and worldwide. Recent outbreaks of Escherichia coli O157:H7 have brought to light the need for improved ways to detect foodborne pathogens and minimize serious outbreaks. Unfortunately, current methods for the detection of foodborne pathogens are time intensive and complex. In this study, we designed a spot immunoassay that uses a UCON-potassium phosphate salt aqueous two-phase system (ATPS) for the preconcentration of O157:H7. This platform was tested with samples of O157:H7 spiked in phosphate-buffered saline and milk. The ATPS was found to improve the detection limit of the spot test, yielding detection at 106 cfu/mL within 30 min. This is the first known application of ATPSs to spot immunoassays. Moreover, detection was successfully achieved without upstream processing or dilution of the sample prior to testing, thereby further simplifying the detection process. This technology's ease of use, sensitivity, and short time to result highlight its potential to advance the spot test as a viable diagnostic tool for foodborne pathogens.

A transferrin variant as the targeting ligand for polymeric nanoparticles incorporated in 3-D PLGA porous scaffolds.

We have developed doxorubicin (DOX)-loaded poly(lactide-co-glycolide) (PLGA) nanoparticles (DP) conjugated with polyethylene glycol (PEG) and transferrin (Tf) to form Tf-PEG-DPs (TPDPs), and incorporated these TPDPs into three-dimensional (3-D) PLGA porous scaffolds to form a controlled delivery system. To our knowledge, this represents the first use of a Tf variant (oxalate Tf) to improve the targeted delivery of drug-encapsulated nanoparticles (NPs) in PLGA scaffolds to PC3 prostate cancer cells. The PLGA scaffolds with TPDPs incorporated have been shown to release drugs for sustained delivery and provided a continuous release of DOX. The MTS assay was also performed to determine the potency of native and oxalate TPDPs, and a 3.0-fold decrease in IC50 values were observed between the native and oxalate TPDPs. The lower IC50 value for the oxalate version signifies greater potency compared to the native version, since a lower concentration of drug was required to achieve the same therapeutic effect. These results suggest that this technology has potential to become a new implantable polymeric device to improve the controlled and targeted drug delivery of Tf-conjugated NPs for cancer therapy.

Mathematical modeling of mutant transferrin-CRM107 molecular conjugates for cancer therapy.

The transferrin (Tf) trafficking pathway is a promising mechanism for use in targeted cancer therapy due to the overexpression of transferrin receptors (TfRs) on cancerous cells. We have previously developed a mathematical model of the Tf/TfR trafficking pathway to improve the efficiency of Tf as a drug carrier. By using diphtheria toxin (DT) as a model toxin, we found that mutating the Tf protein to change its iron release rate improves cellular association and efficacy of the drug. Though this is an improvement upon using wild-type Tf as the targeting ligand, conjugated toxins like DT are unfortunately still highly cytotoxic at off-target sites. In this work, we address this hurdle in cancer research by developing a mathematical model to predict the efficacy and selectivity of Tf conjugates that use an alternative toxin. For this purpose, we have chosen to study a mutant of DT, cross-reacting material 107 (CRM107). First, we developed a mathematical model of the Tf-DT trafficking pathway by extending our Tf/TfR model to include intracellular trafficking via DT and DT receptors. Using this mathematical model, we subsequently investigated the efficacy of several conjugates in cancer cells: DT and CRM107 conjugated to wild-type Tf, as well as to our engineered mutant Tf proteins (K206E/R632A Tf and K206E/R534A Tf). We also investigated the selectivity of mutant Tf-CRM107 against non-neoplastic cells. Through the use of our mathematical model, we predicted that (i) mutant Tf-CRM107 exhibits a greater cytotoxicity than wild-type Tf-CRM107 against cancerous cells, (ii) this improvement was more drastic with CRM107 conjugates than with DT conjugates, and (iii) mutant Tf-CRM107 conjugates were selective against non-neoplastic cells. These predictions were validated with in vitro cytotoxicity experiments, demonstrating that mutant Tf-CRM107 conjugates is indeed a more suitable therapeutic agent. Validation from in vitro experiments also confirmed that such whole-cell kinetic models can be useful in cancer therapeutic design.

Polypeptide-Based Gold Nanoshells for Photothermal Therapy.

Targeted killing of cancer cells by engineered nanoparticles holds great promise for noninvasive photothermal therapy applications. We present the design and generation of a novel class of gold nanoshells with cores composed of self-assembled block copolypeptide vesicles with photothermal properties. Specifically, poly(L-lysine)60- block-poly(L-leucine)20 (K60L20) block copolypeptide vesicles coated with a thin layer of gold demonstrate enhanced absorption of light due to surface plasmon resonance (SPR) in the near-infrared range. We show that the polypeptide-based K60L20 gold nanoshells have low toxicity in the absence of laser exposure, significant heat generation upon exposure to near-infrared light, and, as a result, localized cytotoxicity within the region of laser irradiation in vitro. To gain a better understanding of our gold nanoshells in the context of photothermal therapy, we developed a comprehensive mathematical model for heat transfer and experimentally validated this model by predicting the temperature as a function of time and position in our experimental setup. This model can be used to predict which parameters of our gold nanoshells can be manipulated to improve heat generation for tumor destruction. To our knowledge, our results represent the first ever use of block copolypeptide vesicles as the core material of gold nanoshells.

Engineering A11 Minibody-Conjugated, Polypeptide-Based Gold Nanoshells for Prostate Stem Cell Antigen (PSCA)-Targeted Photothermal Therapy.

Currently, there is no curative treatment for advanced metastatic prostate cancer, and options, such as chemotherapy, are often nonspecific, harming healthy cells and resulting in severe side effects. Attaching targeting ligands to agents used in anticancer therapies has been shown to improve efficacy and reduce nonspecific toxicity. Furthermore, the use of triggered therapies can enable spatial and temporal control over the treatment. Here, we combined an engineered prostate cancer-specific targeting ligand, the A11 minibody, with a novel photothermal therapy agent, polypeptide-based gold nanoshells, which generate heat in response to near-infrared light. We show that the A11 minibody strongly binds to the prostate stem cell antigen that is overexpressed on the surface of metastatic prostate cancer cells. Compared to nonconjugated gold nanoshells, our A11 minibody-conjugated gold nanoshell exhibited significant laser-induced, localized killing of prostate cancer cells in vitro. In addition, we improved upon a comprehensive heat transfer mathematical model that was previously developed by our laboratory. By relaxing some of the assumptions of our earlier model, we were able to generate more accurate predictions for this particular study. Our experimental and theoretical results demonstrate the potential of our novel minibody-conjugated gold nanoshells for metastatic prostate cancer therapy.

Development of quantitative radioactive methodologies on paper to determine important lateral-flow immunoassay parameters.

The lateral-flow immunoassay (LFA) is a well-established diagnostic technology that has recently seen significant advancements due in part to the rapidly expanding fields of paper diagnostics and paper-fluidics. As LFA-based diagnostics become more complex, it becomes increasingly important to quantitatively determine important parameters during the design and evaluation process. However, current experimental methods for determining these parameters have certain limitations when applied to LFA systems. In this work, we describe our novel methods of combining paper and radioactive measurements to determine nanoprobe molarity, the number of antibodies per nanoprobe, and the forward and reverse rate constants for nanoprobe binding to immobilized target on the LFA test line. Using a model LFA system that detects for the presence of the protein transferrin (Tf), we demonstrate the application of our methods, which involve quantitative experimentation and mathematical modeling. We also compare the results of our rate constant experiments with traditional experiments to demonstrate how our methods more appropriately capture the influence of the LFA environment on the binding interaction. Our novel experimental approaches can therefore more efficiently guide the research process for LFA design, leading to more rapid advancement of the field of paper-based diagnostics.

An Aqueous Two-Phase System for the Concentration and Extraction of Proteins from the Interface for Detection Using the Lateral-Flow Immunoassay.

The paper-based immunoassay for point-of-care diagnostics is widely used due to its low cost and portability over traditional lab-based assays. Lateral-flow immunoassay (LFA) is the most well-established paper-based assay since it is rapid and easy to use. However, the disadvantage of LFA is its lack of sensitivity in some cases where a large sample volume is required, limiting its use as a diagnostic tool. To improve the sensitivity of LFA, we previously reported on the concentration of analytes into one of the two bulk phases of an aqueous two-phase system (ATPS) prior to detection. In this study, we preserved the advantages of LFA while significantly improving upon our previous proof-of-concept studies by employing a novel approach of concentrating gold nanoparticles, a common LFA colorimetric indicator. By conjugating specific antibodies and polymers to the surfaces of the particles, these gold nanoprobes (GNPs) were able to capture target proteins in the sample and subsequently be concentrated within 10 min at the interface of an ATPS solution comprised of polyethylene glycol, potassium phosphate, and phosphate-buffered saline. These GNPs were then extracted and applied directly to LFA. By combining this prior ATPS interface extraction with LFA, the detection limit of LFA for a model protein was improved by 100-fold from 1 ng/µL to 0.01 ng/µL. Additionally, we examined the behavior of the ATPS system in fetal bovine serum and synthetic urine to more closely approach real-world applications. Despite using more complex matrices, ATPS interface extraction still improved the detection limit by 100-fold within 15 to 25 min, demonstrating the system's potential to be applied to patient samples.