The lateral-flow immunoassay (LFA) is an inexpensive point-of-care (POC) paper-based diagnostic device with the potential to rapidly detect disease biomarkers in resource-poor settings. Although LFA is inexpensive, easy to use, and requires no laboratory equipment, it is limited by its sensitivity, which remains inferior to that of gold standard laboratory-based assays. Our group is the only one to have previously utilized various aqueous two-phase systems (ATPSs) to enhance LFA detection. In those studies, the sample was concentrated by an ATPS in a test tube and could only be applied to LFA after it had been extracted manually. Here, we bypass the extraction step by seamlessly integrating a polyethylene glycol-potassium phosphate ATPS with downstream LFA detection in a simple, inexpensive, power-free, and portable all-in-one diagnostic device. We discovered a new phenomenon in which the target biomarkers simultaneously concentrate as the ATPS solution flows through the paper membranes, and our device features a 3-D paper well that was designed to exploit this phenomenon. Studies with this device, which were performed at room temperature in under 25 min, demonstrated a 10-fold improvement in the detection limit of a model protein, transferrin. Our next-generation LFA technology is rapid, affordable, easy-to-use, and can be applied to existing LFA products, thereby providing a new platform for revolutionizing the current state of disease diagnosis in resource-poor settings.

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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 Tfconjugated, 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. We are grateful for the insightful comments from the reviewers, and have addressed all of the concerns. A detailed summary of our responses to the comments has been uploaded as the "Lee et al Response to Reviewers" file. IJP AUTHOR CHECKLISTDear Author, It frequently happens that on receipt of an article for publication, we find that certain elements of the manuscript, or related information, is missing. This is regrettable of course since it means there will be a delay in processing the article while we obtain the missing details.In order to avoid such delays in the publication of your article, if accepted, could you please run through the list of items below and make sure you have completed the items. Note that the text bolded in blue represents text that has been added to our original document, and text bolded in red with the strikethrough represents text that has been removed. Overall Manuscript Details Reviewer 11. In the Introduction (Line 55), the author claimed that the commercial DOX liposome technology has limitations, such as instability and short circulation half-life of the vesicles in the body. But this manuscript did not contain any in vitro and in vivo stability data about the Tf-DPEL, such as stability in PBS buffer or plasma. The detailed data of Tf-DPEL should be added in.We thank the reviewer for this point regarding vesicle stability. We have removed this line from the Introduction as this was not within the scope of this paper. Introduction (Line 52-56)DOXIL® is currently FDA approved for treating Kaposi's sarcoma and recurrent ovarian cancer, and is under clinical trials for ...

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