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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.

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.

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.

ObjectiveA 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 designOur 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.SettingTertiary medical center.Subjects and methodsTests 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.ResultsOur 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.ConclusionHerein 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.

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.

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.

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.

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.

We have developed a facile method for preparation of ionic copolypeptide vesicles that requires no protecting groups or expensive components. We prepared amphiphilic copolypeptides containing segments of water-soluble methionine sulfonium residues. These were derived by aqueous phase, direct alkylation of a fully hydrophobic precursor diblock copolypeptide, poly(L-methionine)65-block-poly(L-leucine0.5-stat-L-phenylalanine0.5)20 (M65[L0.5/F0.5]20), with simple alkylating reagents. Methylation of M65(L0.5/F0.5)20 gave the cationic methyl-methionine sulfonium derivative MM65(L0.5/F0.5)20, and carboxymethylation gave the zwitterionic carboxymethyl-methionine sulfonium derivative MC65(L0.5/F0.5)20. Assembly of MM65(L0.5/F0.5)20 or MC65(L0.5/F0.5)20 in water gave rise to vesicles with average diameters of a few microns, which could then be extruded to nanoscale diameters. While the cationic MM-based vesicles were found to be cytotoxic, the zwitterionic MC-based vesicles were found to possess minimal cytotoxicity.

We previously investigated the intracellular trafficking properties of our novel poly(l-glutamate)60-b-poly(l-leucine)20 (E60L20) vesicles (EL vesicles) conjugated to transferrin (Tf). In this study, we expand upon our previous work by investigating the drug encapsulation, release, and efficacy properties of our novel EL vesicles for the first time. After polyethylene glycol (PEG) was conjugated to the vesicles for steric stability, doxorubicin (DOX) was successfully encapsulated in the vesicles using a modified pH-ammonium sulfate gradient method. Tf was subsequently conjugated to the vesicles to provide active targeting to cancer cells and a mode of internalization into the cells. These Tf-conjugated, DOX-loaded, PEGylated EL (Tf-DPEL) vesicles exhibited colloidal stability and were within the allowable size range for passive and active targeting. A mathematical model was then derived to predict drug release from the Tf-DPEL vesicles by considering diffusive and convective mass transfer of DOX. Our mathematical model reasonably predicted our experimentally measured release profile with no fitted parameters, suggesting that the model could be used in the future to manipulate drug carrier properties to alter drug release profiles. Finally, an in vitro cytotoxicity assay was used to demonstrate that the Tf-DPEL vesicles exhibited enhanced drug carrier efficacy in comparison to its non-targeted counterpart.

Cell penetrating peptides (CPPs) are intriguing molecules that have received much attention, both in terms of mechanistic analysis and as transporters for intracellular therapeutic delivery. Most CPPs contain an abundance of cationic charged residues, typically arginine, where the amino acid compositions, rather than specific sequences, tend to determine their ability to enter cells. Hydrophobic residues are often added to cationic sequences to create efficient CPPs, but typically at the penalty of increased cytotoxicity. Here, we examined polypeptides containing glycosylated, cationic derivatives of methionine, where we found these hydrophilic polypeptides to be surprisingly effective as CPPs and to also possess low cytotoxicity. X-ray analysis of how these new polypeptides interact with lipid membranes revealed that the incorporation of sterically demanding hydrophilic cationic groups in polypeptides is an unprecedented new concept for design of potent CPPs.

We prepared dual hydrophilic triblock copolypeptide vesicles that form both micron and nanometer scale vesicles in aqueous media. The incorporation of terminal homoarginine segments into methionine sulfoxide-based vesicles was found to significantly enhance their cellular uptake compared to a non-ionic control. We also demonstrated that diblock and triblock copolypeptides with similar hydrophobic domains were found to mix well and form vesicle populations with uniform compositions. Blending of amphiphiles in vesicle nanocarriers was found to impart these materials with many advantageous properties, including good cellular uptake while maintaining minimal toxicity, as well as biological responsiveness to promote vesicle disruption and release of encapsulated cargos.

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.

ABSTRACT The development of point-of-need (PON) diagnostics for viruses has the potential to prevent pandemics and protects against biological warfare threats. Here we discuss the approach of using aqueous two-phase systems (ATPSs) to concentrate biomolecules prior to the lateral-flow immunoassay (LFA) for improved viral detection. In this paper, we developed a rapid PON detection assay as an extension to our previous proof-of-concept studies which used a micellar ATPS. We present our investigation of a more rapid polymer-salt ATPS that can drastically improve the assay time, and show that the phase containing the concentrated biomolecule can be extracted prior to macroscopic phase separation equilibrium without affecting the measured biomolecule concentration in that phase. We could therefore significantly decrease the time of the diagnostic assay with an early extraction time of just 30 min. Using this rapid ATPS, the model virus bacteriophage M13 was concentrated between approximately 2 and 10-fold by altering the volume ratio between the two phases. As the extracted virus-rich phase contained a high salt concentration which destabilized the colloidal gold indicator used in LFA, we decorated the gold nanoprobes with polyethylene glycol (PEG) to provide steric stabilization, and used these nanoprobes to demonstrate a 10-fold improvement in the LFA detection limit. Lastly, a MATLAB script was used to quantify the LFA results with and without the pre-concentration step. This approach of combining a rapid ATPS with LFA has great potential for PON applications, especially as greater concentration-fold improvements can be achieved by further varying the volume ratio. Biotechnol. Bioeng. 2014;111: 2499-2507. © 2014 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]