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Protein electrophoresis and immunoblotting are indispensable analytical tools for the characterization of proteins and posttranslational modifications in complex sample matrices. Owing to the lack of automation, commonly employed slab-gel systems suffer from high time demand, significant sample/antibody consumption, and limited reproducibility. To overcome these limitations, we developed a paper-based open microfluidic platform for electrophoretic protein separation and subsequent transfer to protein-binding membranes for immunoprobing. Electrophoresis microstructures were digitally printed into cellulose acetate membranes that provide mechanical stability while maintaining full accessibility of the microstructures for consecutive immunological analysis. As a proof-of-concept, we demonstrate separation of fluorescently labeled marker proteins in a wide molecular weight range (15-120 kDa) within only 15 min, reducing the time demand for the entire workflow (from sample preparation to immunoassay) to approximately one hour. Sample consumption was reduced 10- to 150-fold compared to slab-gel systems, owing to system miniaturization. Moreover, we successfully applied the paper-based approach to complex samples such as crude bacterial cell extracts. We envisage that this platform will find its use in protein analysis workflows for scarce and precious samples, providing a unique opportunity to extract profound immunological information from limited sample amounts in a fast fashion with minimal hands-on time.

Proteinuria is a condition in which an excessive amount of protein is excreted in urine. It is, among others, an indicator of kidney disease or risk of cardiovascular disease. Rapid and reliable diagnosis and monitoring of proteinuria is of great importance for both patients and their physicians. For that reason, a paper-based sensor for proteinuria diagnosis was designed, optimized, and validated utilizing smartphone-assisted signal acquisition. In the first step, a few commonly employed protein assays were optimized and compared in terms of analytical performance on paper matrix. The tetrabromophenol blue method was selected as the one providing a sufficiently low limit of detection (39 mg·L−1) on the one hand and appropriate long-term stability (up to 3 months) on the other hand. The optimized assay was employed for protein-to-creatinine ratio (PCR) determination on a single paper-based sensor. For both analytes the linear ranges were within the clinically relevant range. The analytical usefulness of the developed sensors was demonstrated by a PCR recovery study in artificial urine. The obtained PCR recoveries were from ca. 80 to 150%.

Sensors based on fluorogenic RNA aptamers have emerged in recent years. These sensors have been used for in vitro and intracellular detection of a broad range of biological and medical targets. However, the potential application of fluorogenic RNA-based sensors for point-of-care testing is still little studied. Here, we report a paper substrate-based portable fluorogenic RNA sensor system. Target detection can be simply performed by rehydration of RNA sensor-embedded filter papers. This affordable sensor system can be used for the selective, sensitive, and rapid detection of different target analytes, such as antibiotics and cellular signaling molecules. We believe that these paper-based fluorogenic RNA sensors show great potential for point-of-care testing of a wide range of targets from small molecules, nucleic acids, proteins, to various pathogens.

Background Brucellosis, as a serious zoonotic infectious disease, has been recognized as a re-emerging disease in the developing countries worldwide. In china, the incidence of brucellosis is increasing each year, seriously threatening the health of humans as well as animal populations. Despite a quite number of diagnostic methods currently being used for brucellosis, innovative technologies are still needed for its rapid and accurate diagnosis, especially in area where traditional diagnostic is unavailable. Methodology/Principal findings In this study, a total of 22 B cell linear epitopes were predicted from five Brucella outer membrane proteins (OMPs) using an immunoinformatic approach. These epitopes were then chemically synthesized, and with the method of indirect ELISA (iELISA), each of them displayed a certain degree of capability in identifying human brucellosis positive sera. Subsequently, a fusion protein consisting of the 22 predicted epitopes was prokaryotically expressed and used as diagnostic antigen in a newly established brucellosis testing method, nano-ZnO modified paper-based ELISA (nano-p-ELISA). According to the verifying test using a collection of sera collected from brucellosis and non-brucellosis patients, the sensitivity and specificity of multiepitope based nano-p-ELISA were 92.38% and 98.35% respectively. The positive predictive value was 98.26% and the negative predictive value was 91.67%. The multiepitope based fusion protein also displayed significantly higher specificity than Brucella lipopolysaccharide (LPS) antigen. Conclusions B cell epitopes are important candidates for serologically testing brucellosis. Multiepitope fusion protein based nano-p-ELISA displayed significantly sensitivity and specificity compared to Brucella LPS antigen. The strategy applied in this study will be helpful to develop rapid and accurate diagnostic method for brucellosis in human as well as animal populations., Author summary Brucellosis is one of the most important zoonosis in the world and has caused tremendous economic losses in agriculture and animal husbandry in many countries. Developing rapid, sensitive and specific diagnostic methods is very important for early detection and treatment of brucellosis patients. In this study, a novel diagnostic technique, nano-ZnO modified paper ELISA, was established. The antigen used in this technique was a fusion protein containing multiple B cell epitopes, which were predicted from Brucella major outer membrane proteins such as Bp26, Omp31, Omp16, Omp2b and Omp25. Comparing to traditional LPS antigen, this multiepitope based antigen displayed considerably higher sensitivity and higher specificity in laboratory. With the strategy described in this paper, more efficient epitopes and protein antigen can be identified in the future. Currently, LPS antigen is only prepared from live Brucella, while protein antigen can be produced in large quantities in prokaryotic expression system. In addition to nano-p-ELISA, this protein antigen can also be used for development other methods such as fluorescent polarization assay (FPA) and immunochromatographic assay (ICA) to meet the varied demand for brucellosis testing.

Integration and storage of bioactive reagents is an important and challenging task in microfluidic paper-based analytical devices (μPADs). Here, we developed a convenient and universal method to store proteins and preserve their activities in μPADs by using aqueous two-phase systems (ATPs) evolved film. A polyethylene glycol (PEG)-dextran (DEX) double-layer film was formed through dehydration of ATPs. Functional biomolecules were stored in the bottom DEX layer on the basis of the biased partitioning and rehydrated conveniently by simple addition of buffer solution at usage. As a demonstration, enzyme immunoassay (EIA) of carcinoembryonic antigen was performed successfully on μPAD integrated with antibodies. Even after 104 days of storage at 4 °C and ambient conditions, the EIA signal just lost less than 10% and 30%, which meet the storage requirements of invitro diagnosis reagents. The ATPs evolved double-layer film has double functions of stabilization and insulation, and provide a high efficiency of biomolecule preservation, thereby promoting the applications of μPADs in POC diagnostic assay.

This work presents an annotation tool that automatically locates mentions of particular amino-acid residues in published papers and identifies the protein concerned. These matches can be provided in context or in a searchable format in order for researchers to better use the existing and future literature.

Isoelectric focusing plays a critical role in the analysis of complex protein samples. Conventionally, isoelectric focusing is implemented with carrier ampholytes in capillary or immobilized pH gradient gel. In this study, we successfully exhibited a carrier ampholyte-free isoelectric focusing on paper-based analytical device. Proof of the concept was visually demonstrated with color model proteins. Experimental results showed that not only a pH gradient was well established along the open paper fluidic channel as confirmed by pH indicator strip, the pH gradient range could also be tuned by the catholyte or anolyte. Furthermore, the isoelectric focusing fractions from the paper channel can be directly cut and recovered into solutions for post analysis with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry. This paper-based isoelectric focusing method is fast, cheap, simple and easy to operate, and could potentially be used as a cost-effective protein sample clean-up method for target protein analysis with mass spectrometry.

We describe the design and characteristics of a paper-based analytical device for analyte concentration enrichment. The device, called a hybrid paper-based analytical device (hyPAD), uses faradaic electrochemistry to create an ion depletion zone (IDZ), and hence a local electric field, within a nitrocellulose flow channel. Charged analytes are concentrated near the IDZ when their electrophoretic and electroosmotic velocities balance. This process is called faradaic ion concentration polarization. The hyPAD is simple to construct and uses only low-cost materials. The hyPAD can be tuned for optimal performance by adjusting the applied voltage or changing the electrode design. Moreover, the throughput of hyPAD is 2 orders of magnitude higher than that of conventional, micron-scale microfluidic devices. The hyPAD is able to concentrate a range of analytes, including small molecules, DNA, proteins, and nanoparticles, in the range of 200−500-fold within 5 min.

[cat] La teoria més plausible de l’evolució del càncer de mama és la que considera el carcinoma ductal in situ (DCIS) com a precursor no obligat del carcinoma ductal invasiu (IDC), que eventualment progressa cap a l’etapa metastàtica. El DCIS es caracteritza per la proliferació de les cèl·lules epitelials confinades dins del lumen dels ductes, sense arribar a travessar la capa de cèl·lules mioepitelials i sense arribar a envair l’estroma mamari, quan aquests fets es produeixen és quan ja es considera IDC. Avui en dia es desconeix què desencadena l’evolució del DCIS i no és possible predir quins casos progressaran ràpidament cap a IDC, i quins d’ells es mantindran estables. Per tant, és imprescindible millorar el coneixement sobre aquestes lesions per predir el risc de progressió i realitzar un tractament a mida en cada cas. En aquest context, cal destacar la influència del microambient com a element clau en l’evolució de DCIS. En aquest estudi s’ha volgut posar especialment atenció en factors neuronals, els quals han estat relacionats amb l’inici, la progressió i la supervivència del càncer de mama. Per aquest motiu, aquesta tesi ha tingut com objectiu l’estudi de dos elements relacionats amb el sistema nerviós com són la SEMA3F i NTN1, per conèixer el seu paper en la transició del DCIS a malaltia invasiva. D'aquesta manera s'ha demostrat que SEMA3F és un element protumoral que és sintetitzat per les cèl·lules epitelials tumorals i que potencia la transició de DCIS a IDC, i s’ha demostrat que almenys parcialment aquests efectes són realitzats a través dels seus correceptors NRP1 i NRP2. A més a més, presenta un efecte protumorogènic en les cèl·lules mioepitelials, afectant-les i modificant-les cap a un fenotip alterat pel tumor. Tanmateix, sembla que el paper antitumoral de les cèl·lules mioepitelials és capaç de contrarestar els efectes de la SEMA3F. De la mateixa manera, s'ha demostrat que NTN1 inhibeix la capacitat d'invasió de les cèl·lules epitelials en IDC, revertint el seu fenotip cap a un de menys agressiu. A més, la sobreexpressió de NTN1 protegeix les cèl·lules mioepitelials enfront de la pèrdua de la seva funció antitumoral. Per últim, s'ha demostrat l'existència de relació entre aquestes dues molècules i el seu rol en DCIS, en tant que la sobreexpressió de SEMA3F modula a la baixa l'expressió de NTN1. En conclusió, aquesta tesi contribueix a un major coneixement del DCIS, i a la seva possible evolució a patologia invasiva, tenint en compte els efectes dels factors neuronals, la SEMA3F com a molècula pro-invasiva, i NTN1 com a element que bloqueja l’evolució a càncer de mama invasiu. Aquest major coneixement és a l’hora una aportació de possibles biomarcadors de diagnòstic i pronòstic del DCIS, i també una proposta de possibles noves dianes terapèutiques per controlar el càncer de mama en la fase pre-invasiva., [eng] The most plausible theory about the evolution of breast cancer considers ductal carcinoma in situ (DCIS) as a non-obligated precursor of the invasive phase (IDC), which eventually ends in a metastatic state. DCIS is characterized by the proliferation of mammary epithelial cells confined to breast ductal structures and surrounded by both, the myoepithelial cell layer and by the basement membrane (BM), without invading the extracellular matrix. From the moment invasion occurs it is considered an IDC. It is unknown what triggers the evolution of DCIS and it is not possible to predict which cases will progress rapidly to IDC and which of them will remain stable. Therefore, it is essential to improve our knowledge about these pre-invasive BC lesions to predict the individual risk of progression and to carry out a tailored treatment in each case. In this context, it is noteworthy that the influence of the microenvironment is key in the evolution of DCIS to IDC. This study was designed to focus on neural factors, which have been previously linked to the onset, progression and survival of breast cancer. For this reason, the aim of this thesis has been to decipher the role of two nervous system-related factors, SEMA3F and NTN1, in the transition from DCIS to invasive disease. On one hand, this thesis shown that SEMA3F is a protumoral element that is synthesized by tumor epithelial cells and promotes the transition from DCIS to IDC at least partially through its coreceptors NRP1 and NRP2. In addition, SEMA3F has a protumorigenic effect on myoepithelial cells, affecting and modifying them towards a tumor-altered phenotype. However, the antitumor role of myoepithelial cells is able to counteract the effects of SEMA3F. On the other hand, NTN1 is shown to inhibit the invasion capacity of epithelial cells in IDC, leading them to a less aggressive tumoral phenotype. In addition, overexpression of NTN1 protects myoepithelial cells against loss of their antitumor function. Finally, evidences are given for the existence of a relationship between these two molecules and their role in DCIS, while overexpression of SEMA3F down-regulates NTN1 expression. In conclusion, this thesis contributes to a better understanding of DCIS and its possible evolution into invasive pathology, considering the effects of neuronal factors, SEMA3F as a pro- invasive molecule and NTN1 as an element that blocks the progression to invasive breast cancer. This deeper knowledge about BC biology is at the same time a contribution to possible biomarkers of diagnosis and prognosis of DCIS, and also a proposal of new targeted treatment strategies to control breast cancer in the more therapeutically affordable pre-invasive phase.

[cat] Partint dels resultats previs obtinguts per aquest grup d’investigació, es planteja la següent hipòtesi de treball: “La proteïna p27 és un regulador de la via de Notch. Aquesta regulació es realitzaria, principalment, controlant la transcripció de diferents components d’aquesta ruta de senyalització, com per exemple el lli-gand Jag1 o el receptor Notch2. Tanmateix, es planteja que p27 podria regular l’expressió d’altres components d’aquesta via”. L’objectiu general d’aquesta tesi és validar aquesta hipòtesi de treball en diferents línies cel·lulars. Per tal de fer-ho, s’han establert els se-güents objectius concrets: 1. Validar els resultats previs obtinguts en un microarray d’expressió realitzats en cèl·lules MEF deficients en p27 vs. cèl·lules MEF control. En aquest microarray s’observava que els les cèl·lules MEFp27KO tenien nivells mes alts de mRNA de diferents components de la via de Notch respecte als controls. 2. Analitzar si la pèrdua de p27 indueix també un increment dels nivells de proteïna dels diferents components de la via de Notch. 3. Generar a partir de cèl·lules de càncer colorectal HCT116, clons p27KO mitjançant la tècnica CRISPR/CAS9 i analitzar en aquestes cèl·lules l'expressió de diferents components de la via de Notch, tant a nivell de mRNA com de proteïna. 4. Analitzar el paper de p27 en l’expressió, tant a nivell de mRNA com de proteïna, de diferents elements de la via de Notch en lí-nies cel·lulars derivades de leucèmies. 5. Estudi del paper dels complexes E2F4/p130 i CDK-Ciclina en la regulació de la via de Notch, seguint el model pre-establert en aquest grup. 6. Identificar quins dels components d’aquesta via estan directa-ment regulats per p27. 7. Establir els mecanismes moleculars que participen en aquesta regulació.

Rocky desertification is a bottleneck that reduces ecological and environmental security in karst areas. Paper mulberry, a unique deciduous tree, shows good performance in rocky desertification areas. Its resistance mechanisms are therefore of high interest. In this study, a lysine acetylation proteomics analysis of paper mulberry seedling leaves was conducted in combination with the purification of acetylated protein by high-precision nano LC-MS/MS. We identified a total of 7130 acetylation sites in 3179 proteins. Analysis of the modified sites showed a predominance of nine motifs. Six positively charged residues: lysine (K), arginine (R), and histidine (H), serine (S), threonine (T), and tyrosine (Y) occurred most frequently at the +1 position, phenylalanine (F) was both detected both upstream and downstream of the acetylated lysines; and the sequence logos showed a strong preference for lysine and arginine around acetylated lysines. Functional annotation revealed that the identified enzymes were mainly involved in translation, transcription, ribosomal structure and biological processes, showing that lysine acetylation can regulate various aspects of primary carbon and nitrogen metabolism and secondary metabolism. Acetylated proteins were enriched in the chloroplast, cytoplasm, and nucleus, and many stress response-related proteins were also discovered to be acetylated, including PAL, HSP70, and ERF. HSP70, an important protein involved in plant abiotic and disease stress responses, was identified in paper mulberry, although it is rarely found in woody plants. This may be further examined in research in other plants and could explain the good adaptation of paper mulberry to the karst environment. However, these hypotheses require further verification. Our data can provide a new starting point for the further analysis of the acetylation function in paper mulberry and other plants.

Proteinoids – thermal proteins – are produced by heating amino acids to their melting point and initiation of polymerisation to produce polymeric chains. Proteinoids swell in aqueous solution into hollow microspheres. The proteinoid microspheres produce endogenous burst of electrical potential spikes and change patterns of their electrical activity in response to illumination. The microspheres can interconnect by pores and tubes and form networks with a programmable growth. We speculate on how ensembles of the proteinoid microspheres can be developed into unconventional computing devices.

Proteinaceous toxins are harmful proteins derived from plants, bacteria, and other natural sources. They pose a risk to human health due to infection and also as possible biological warfare agents. Paper spray mass spectrometry (PS-MS) with wipe sampling was used to detect proteins from surfaces as a potential tool for identifying the presence of these toxins. Proteins ranging in mass between 12.4 and 66.5 kDa were tested, including a biological toxin simulant/vaccine for Staphylococcal enterotoxin B (SEBv). Various substrates were tested for these representative proteins, including a laboratory bench, a notebook cover, steel, glass, plant leaf and vinyl flooring. Carbon sputtered porous polyethylene (CSPP) was found to outperform typical chromatography paper used for paper spray, as well as carbon nanotube (CNT)-coated paper and polyethylene (PE), which have been previously shown to be well-suited for protein analysis. Low microgram quantities of the protein toxin simulant and other test proteins were successfully detected with good signal-to-noise from surfaces using a porous wipe. These applications demonstrate that PS-MS can potentially be used for rapid, sample preparation-free detection of proteins and biological warfare agents, which would be beneficial to first responders and warfighters.

Paper Spray Ionization (PSI) is commonly applied for the analysis of small molecules, including drugs, metabolites, and pesticides in biological fluids, due to its high versatility, simplicity, and low costs. In this study, a new setup called Solvent Assisted Paper Spray Ionization (SAPSI), able to increase data acquisition time, signal stability, and repeatability, is proposed to overcome common PSI drawbacks. The setup relies on an integrated solution to provide ionization potential and constant solvent flow to the paper tip. Specifically, the ion source was connected to the instrument fluidics along with the voltage supply systems, ensuring a close control over the ionization conditions. SAPSI was successfully applied for the analysis of different classes of biomolecules: amyloidogenic peptides, proteins, and N-glycans. The prolonged analysis time allowed real-time monitoring of processes taking places on the paper tip, such as amyloid peptides aggregation and disaggregation phenomena. The enhanced signal stability allowed to discriminate protein species characterized by different post translational modifications and adducts with electrophilic compounds, both in aqueous solutions and in biofluids, such as serum and cerebrospinal fluid, without any sample pretreatment. In the next future, application to clinical relevant modifications, could lead to the development of quick and cost-effective diagnostic tools.

Current analytical methods, either point-of-care or centralized detection, are not able to meet recent demands of patient-friendly testing and increased reliability of results. Here, we describe a two-point separation on-demand diagnostic strategy based on a paper-based mass spectrometry immunoassay platform that adopts stable and cleavable ionic probes as mass reporter; these probes make possible sensitive, interruptible, storable, and restorable on-demand detection. In addition, a new touch paper spray method was developed for on-chip, sensitive, and cost-effective analyte detection. This concept is successfully demonstrated via (i) the detection of Plasmodium falciparum histidine-rich protein 2 antigen and (ii) multiplexed and simultaneous detection of cancer antigen 125 and carcinoembryonic antigen.

Paper-based fluorescence resonance energy transfer assay (FRET) is gaining great interest in detecting macro-biological molecule. It is difficult to achieve conveniently and fast detection for macro-biological molecule. Herein, a graphene oxide (GO)-based paper chip (glass fiber) integrated with fluorescence labeled single-stranded DNA (ssDNA) for fast, inexpensive and direct detection of biological macromolecules (proteins and nucleic acids) has been developed. In this paper, we employed the Cy3/FAM-labeled ssDNA as the reporter and the GO as quencher and the original glass fiber paper as data acquisition substrates. The chip which was designed and fabricated by a cutting machine is a miniature biosensor that monitors fluorescence recovery from resonance energy transfer. The hybridization assays and fluorescence detection were all simplified, and the surface of the chip did not require immobilization or washing. A Nikon Eclipse was employed as excited resource and a commercial digital camera was employed for capturing digital images. This paper-based microfluidics chip has been applied in the detection of proteins and nucleic acids. The biosensing capability meets many potential requirements for disease diagnosis and biological analysis.

This paper describes the need for technologies that improve analytical sensitivity to proteins to better define and monitor the progression from heath to disease over the course of an individual's life. These technologies have the potential to allow the early diagnosis of disease, and trigger treatments at the time when they have the greatest opportunity to be effective. We will describe a technology that we have developed for high sensitivity protein detection, namely, single molecule arrays (Simoa). Simoa is based on the capture of protein molecules on magnetic beads, labeling each protein with an enzyme, and counting of single enzyme labels on beads that are isolated in arrays of femtoliter wells. Simoa has enabled the detection of proteins at subfemtomolar concentrations in a variety of biological fluids. We describe the impact of higher sensitivity of proteins using Simoa on: less invasive testing; earlier detection of disease; providing biomarker baseline profiles for healthy individuals; testing of small sample volumes; monitoring of therapeutic efficacy; faster tests; and detection of proteins in complex samples. We also provide a perspective of how new technologies that allow the low-cost manufacture and miniaturization of Simoa could drive the next wave of analytical devices, including wearables.

To ensure protein beverage safety and prevent illegal melamine use to artificially increase protein content, a rapid, on-site, ultrasensitive detection method for melamine must be developed because melamine is detrimental to human health. Herein, an ultrasensitive time-resolved fluorescence detection paper (TFDP) was developed to detect melamine in protein beverages within 15 min using a one-step sample preparation. The lower limits of detection were 0.89, 0.94, and 1.05 ng/mL, and the linear ranges were 2.67–150, 2.82–150, and 3.15–150 ng/mL (R2 > 0.982) for peanut, walnut, and coconut beverages, respectively. The recovery rates were 85.86–110.60% with a coefficient of variation

In this work, a disposable paper-plastic hybrid microfluidic lab-on-a-chip (LOC) has been developed and successfully applied for the colorimetric measurement of urine by the smartphone-based optical platform using a "UrineAnalysis" Android app. The developed device was cost-effectively implemented as a stand-alone hybrid LOC by incorporating the paper-based conventional reagent test strip inside the plastic-based LOC microchannel. The LOC device quantitatively investigated the small volume (40 μL) of urine analytes for the colorimetric reaction of glucose, protein, pH, and red blood cell (RBC) in integration with the finger-actuating micropump. On the basis of our experiments, the conventional urine strip showed large deviation as the reaction time goes by, because dipping the strip sensor in a bottle of urine could not control the reaction volume. By integrating the strip sensor in the LOC device for urine analysis, our device significantly improves the time-dependent inconstancy of the conventional dipstick-based urine strip, and the smartphone app used for image analysis enhances the visual assessment of the test strip, which is a major user concern for the colorimetric analysis in point-of-care (POC) applications. As a result, the user-friendly LOC, which is successfully implemented in a disposable format with the smartphone-based optical platform, may be applicable as an effective tool for rapid and qualitative POC urinalysis.

H1 NMR relaxometry is a method that is extremely sensitive to the presence of magnetic nanoparticles, which significantly affect the transverse relaxation time of the water proton. Accordingly, the use of magnetic nanoparticles as labels allows detection of even extremely small amounts of the test substance. This paper analyzes the prospects for applying the method of solid-phase NMR-relaxometric determination of biologically active molecules. The nitrocellulose membranes are chosen as a solid phase and nanoparticles based on iron core with a carbon shell are used as magnetic labels. The possibility of detecting small concentrations of magnetic particles in porous medium is demonstrated. Finally, the ability to detect extremely low concentrations of an analyte, in this case, streptavidin protein (0.5 ng/ml to 100 ng/ml), which is actively used in various fields of biology and medicine, is demonstrated. © Published under licence by IOP Publishing Ltd. Russian Science Foundation, RSF: 17-15-01116 The work was carried out within the Russian Science Foundation project 17-15-01116. equipment of the Ural Center for Shared Use Modern nanotechnology UrFU was used.

This work describes a device for electrochemical enzyme-linked immunosorbent assay (ELISA) designed for low-resource settings and diagnostics at the point of care. The device is fabricated entirely in hydrophobic paper, produced by silanization of paper with decyl trichlorosilane, and comprises two zones separated by a central crease: an embossed microwell, on the surface of which the antigen or antibody immobilization and recognition events occur, and a detection zone where the electrodes are printed. The two zones are brought in contact by folding the device along this central crease; the analytical signal is recorded from the folded configuration. Two proof-of-concept applications, an electrochemical direct ELISA for the detection of rabbit IgG as a model antigen in buffer and an electrochemical sandwich ELISA for the detection of malarial histidine-rich protein from Plasmodium falciparum (Pf HRP2) in spiked human serum, show the versatility of this device. The limit of detection of the electrochemical sandwich ELISA for the quantification of Pf HRP2 in spiked human serum was 4 ng mL(-1) (10(2) pmol L(-1)), a value within the range of clinically relevant concentrations.

Although microfluidic approach is widely used in various point of care diagnostics, its implementation in surface enhanced Raman spectroscopy (SERS)-based detection is challenging. This is because SERS signal depends on plasmonic nanoparticle aggregation induced generation of stable electromagnetic hot spots and in currently available microfluidic platform this condition is difficult to adapt. Here we show that SERS can be adapted using simple paper based microfluidic system where both the plasmonic nanomaterials and analyte are used in mobile phase. This approach allows analyte induced controlled particle aggregation and electromagnetic hot spot generation inside the microfluidic channel with the resultant SERS signal, which is highly reproducible and sensitive. This approach has been used for reproducible detection of protein in the pico to femtomolar concentration. Presented approach is simple, rapid, and cost-effective, and requires low sample volume. Method can be extended for SERS-based detection of other biomolecules.

A concept integrating sampling and protein digestion is introduced here combining fast and simple fabrication by wax printing on filter paper with trypsin immobilized polymer beads. The paper reactors showed promising results with a high degree of protein digestion within fifty minutes in model protein mixtures as well as in human blood. The model protein mixture was used for the evaluation of performance both with and without a reduction and alkylation step. The paper reactors without reduction and alkylation showed between 46% and 75% protein sequence coverage and between five and 20 high confidence peptides (one and five zero missed cleavage peptides, respectively). Compared to a conventional in-solution approach, the paper reactor showed 10% less protein sequence coverage, 29% fewer high confidence peptides and 19% fewer high confidence peptides with zero missed cleavages. Placement of the protein reduction and alkylation step (before or after protein digestion) was shown to be of low importance. The storage stability of the paper reactors with (six weeks) and without (twelve weeks) tryptic peptides was satisfactory. The ability of the paper reactors to digest complex biological samples was investigated by comparison with human whole blood samples prepared using a conventional dried blood spot (DBS) procedure with overnight digestion in non-targeted analysis. The reactors showed a comparable performance with 75 ± 25 for the protein groups compared to 76 ± 5 for the DBS samples. Additionally, 267 ± 72 and 335 ± 11 unique peptides (high confidence) were identified for on-paper digestion and DBS, respectively.

[cat] L’obesitat s’ha convertit en les últimes dècades en un greu problema per la salut pública arribant a ser una de les principals causes de morbiditat, incapacitació i mortalitat prematura. Un teixit que pot tenir un paper important en la protecció contra l’obesitat és el teixit adipós marró (TAM). El TAM és un dels principals teixits consumidors d’energia en els mamífers. El TAM, descrit primerament en rosegadors i en nounats ha anat guanyant rellevància la última dècada amb el descobriment de la presència d’aquest i la seva activació front a estímuls de fred en humans adults. Recentment, s’ha descrit un paper secretor del TAM. En resposta a l’activació termogènica el TAM allibera factors que actuen de manera autocrina, paracrina o endocrina reforçant aquesta activació. A aquests factors secretats pel TAM se’ls anomena adipoquines marrons o batoquines. Ara fa aproximadament uns 20 anys sis laboratoris van publicar en poc temps la descripció d’un nou factor secretable de la família del TGFβ, anomenat GDF15. GDF15 és coneguda principalment com una citocina de resposta a estrès. La seva expressió augmenta en resposta tant a situacions d’estrès patològic, com gran varietat de malalties, com en situacions d’estrès fisiològiques com ara durant la gestació. Recentment, s’ha descobert un receptor de GDF15 al cervell, GFRAL. S’ha vist que GDF15 actua a nivell central com a factor anorexigènic. Tot i això, altres estudis postulen que GDF15 té altres accions en teixits perifèrics. En aquesta tesi, s’ha identificat el factor GDF15 com una adipoquina marró o batoquina, alliberada pels adipòcits marrons i beix en resposta a l’activació termogènica d’aquestes cèl·lules. S’ha determinat la via adrenèrgica β3 com la responsable de l’augment de l’expressió i secreció de GDF15 en resposta a l’estímul termogènic, i s’han trobat implicades proteïnes d’aquesta via com la PKA i adipoquines marrons prèviament identificades, com FGF21. A més a més, s’ha observat que el GDF15 produït pels adipòcits té afectes en els macròfags residents dins del teixit, reprimint-ne la inflamació. Aquests resultants suggereixen que la secreció de GDF15 per part dels adipòcits marrons pot ser un mecanisme per regular localment l’activitat pro-inflamatòria dels macròfags associada amb una millora de l’activitat del teixit adipós marró. Paral·lelament, s’han realitzat diferents estudis sobre GDF15 en poblacions humanes. S’ha vist com GDF15 augmenta en patologia mitocondrial en nens i s’han determinat els miotubs com les cèl·lules responsables d’aquest augment. També s’han analitzat els nivells circulants de GDF15 durant el desenvolupament postnatal, observant una possible associació entre aquests nivells i el pes dels nounats en el moment del naixement. Per últim, s’ha avaluat la resposta de GDF15 a un exercici intens; s’ha vist un dràstic augment de GDF15 circulant en atletes no professionals en acabar la marató de Barcelona del 2017, que era totalment revertit dos dies després de la realització de la cursa. En més d’un dels estudis s’ha observat un paral·lelisme entre els nivells de GDF15 i els nivells d’FGF21, tot i això sembla ser que aquestes dues proteïnes que sovint augmenten en paral·lel actuen a través de vies totalment independents. En resum, en aquesta tesi hem estudiat la proteïna GDF15 en situacions molt diverses; patològiques i fisiològiques, sistèmiques i locals i en models humans i de ratolí. GDF15 sembla ser una important proteïna reguladora del metabolisme, amb efectes molt diversos en funció de teixit i condició. Regula la termogènesi localment en el teixit adipós, esta implicada en el desenvolupament post-natal actuant a nivell central i respon a situacions d’estrès tant patològiques (patologia mitocondrial) com fisiològiques (exercici). És necessari, però, seguir investigant per tal d’entendre els mecanismes i la importància biològica de GDF15 en cadascuna d’aquestes situacions., [eng] Brown adipose tissue (BAT) is the main site of energy expenditure in mammals, via adaptive thermogenesis. The thermogenic capacity of BAT occurs through the unique expression of the uncoupling protein UCP1 and high oxidative capacity of brown adipocytes. High BAT activity favors a negative energy balance, protecting against obesity. GDF15 (growth and differentiation factor-15) is a member of the TGFβ superfamily and is secreted by the liver, placenta and other organs. Its biological function is unclear, but it has been proposed as a biomarker of various pathological states, such as inflammatory diseases, cardiovascular disease, or obesity. Recently, several studies reported the presence of a specific receptor for GDF15 in the brain, known as GFRAL, which mediates the anorexigenic response to this molecule. In this thesis we were able to analyze this protein in different mouse and human models and in different physiological and pathological conditions. In mice, cold exposure strongly induced GDF15 expression in BAT, norepinephrine and cAMP induced GDF15 expression and release by cells through protein kinase A-mediated mechanisms and the GDF15 released by brown adipocytes targeted macrophages, and down-regulated the expression of pro-inflammatory genes. These results indicated that GDF15 is a novel brown adipokine released by brown adipocytes in response to thermogenic activation. In humans, children with mitochondrial diseases have increased GDF15 levels compared to healthy controls, proposing GDF15 as a biomarker for these diseases. GDF15, as well as FGF21, mRNA expression and protein secretion, were significantly induced after treatment of myotubes with drugs that block the respiratory chain, indicating that myotubes are the responsible cells for GDF15 production in these conditions. In another study, GDF15 levels appeared significantly high at birth and dropped at age 4 months, particularly in infants born with lower weight, and continued to decline progressively in both subgroups reaching adult concentrations by age 24 months. Finally, we assessed the value of GDF15 and FGF21 as biomarkers in relation to the biological response to strenuous exercise. A marked increase in the mean levels of GDF15 and FGF21 was found immediately after a marathon race. These levels returned to basal values 48h post-race.

The design of appropriate diagnostic assays for the point of care requires development of suitable biosensors, detection methods, and diagnostic platforms for sensitive, quantitative detection of biological analytes. Protein targets in particular are especially challenging to detect quantitatively and sensitively due to the lack of amplification strategies akin to nucleic acid amplification. However, recent advances in transducer and biosensor design, new detection labels, and paper-based microfluidics may realize the goal of sensitive, fast, portable, and low-cost protein detection. In this review, we discuss the biochemistry, optics, and engineering advances that may be leveraged to design such a sensitive protein diagnostic assay. The binding kinetics, mechanisms of binding in porous networks, and potential transducers are explained in detail. We discuss the relative merits of various optical detection strategies, potential detection labels, optical readout approaches, and image-processing techniques that are amenable to point-of-care use. To conclude, we present a systematic analysis of potential approaches to enhance the sensitivity of paper-based assays. The assay development framework presented here provides bioassay developers a strategy to methodically enhance the sensitivity and point-of-care suitability of protein diagnostics.

The first step in curing a disease is being able to detect the disease effectively. Paper-based microfluidic devices are biodegradable and can make diagnosing diseases cost-effective and easy in almost all environments. We created a three-dimesnional (3D) paper device using wax printing fabrication technique and basic principles of origami. This design allows for a versatile fabrication technique over previously reported patterning of SU-8 photoresist on chromatography paper by employing a readily available wax printer. The design also utilizes multiple colorimetric assays that can accommodate one or more analytes including urine, blood, and saliva. In this case to demonstrate the functionality of the 3D paper-based microfluidic system, a urinalysis of protein and glucose assays is conducted. The amounts of glucose and protein introduced to the device are found to be proportional to the color change of each assay. This color change was quantified by use of Adobe Photoshop. Urine samples from participants with no pre-existing health conditions and one person with diabetes were collected and compared against synthetic urine samples with predetermined glucose and protein levels. Utilizing this method, we were able to confirm that both protein and glucose levels were in fact within healthy ranges for healthy participants. For the participant with diabetes, glucose was found to be above the healthy range while the protein level was in the healthy range.

Biosensors have emerged as a valuable tool with high specificity and sensitivity for fast and reliable detection of hazardous substances in drinking water. Numerous substances have been addressed using synthetic biology approaches. However, many proposed biosensors are based on living, genetically modified organisms and are therefore limited in shelf life, usability and biosafety. We addressed these issues by the construction of an extensible, cell-free biosensor. Storage is possible through freeze drying on paper. Following the addition of an aqueous sample, a highly efficient cell-free protein synthesis (CFPS) reaction is initiated. Specific allosteric transcription factors modulate the expression of 'superfolder' green fluorescent protein (sfGFP) depending on the presence of the substance of interest. The resulting fluorescence intensities are analyzed with a conventional smartphone accompanied by simple and cheap light filters. An ordinary differential equitation (ODE) model of the biosensors was developed, which enabled prediction and optimization of performance. With an optimized cell-free biosensor based on the Shigella flexneri MerR transcriptional activator, detection of 6 mug/L Hg(II) ions in water was achieved. Furthermore, a completely new biosensor for the detection of gamma-hydroxybutyrate (GHB), a substance used as date-rape drug, was established by employing the naturally occurring transcriptional repressor BlcR from Agrobacterium tumefaciens.

We have demonstrated a multistep 2-dimensional paper network immunoassay based on controlled rehydration of patterned, dried reagents. Previous work has shown that signal enhancement improves the limit of detection in 2-dimensional paper network assays, but until now, reagents have only been included as wet or dried in separate conjugate pads placed at the upstream end of the assay device. Wet reagents are not ideal for point-of-care because they must be refrigerated and typically limit automation and require more user steps. Conjugate pads allow drying but do not offer any control of the reagent distribution upon rehydration and can be a source of error when pads do not contact the assay membrane uniformly. Furthermore, each reagent is dried on a separate pad, increasing the fabrication complexity when implementing multistep assays that require several different reagents. Conversely, our novel method allows for consistent, controlled rehydration from patterned reagent storage depots directly within the paper membrane. In this assay demonstration, four separate reagents were patterned in different regions of the assay device: a gold-antibody conjugate used for antigen detection and three different signal enhancement components that must not be mixed until immediately before use. To show the viability of patterning and drying reagents directly onto a paper device for dry reagent storage and subsequent controlled release, we tested this device with the malaria antigen Plasmodium falciparum histidine-rich protein 2 (PfHRP2) as an example of target analyte. In this demonstration, the signal enhancement step increases the visible signal by roughly 3-fold and decreases the analytical limit of detection by 2.75-fold.

Pickering high internal phase emulsions (HIPEs) are normally highly concentrated emulsions stabilized by colloidal particles with a minimum internal phase volume fraction of 0.74. They have received considerable attention in many fields, including pharmaceuticals, tissue engineering, foods, and personal care products. The aim of this perspective is to update the current knowledge on the field of protein-based Pickering HIPEs, emphasizing those aspects that need to be explored and clarified. Research progress in constructing HIPEs by protein-type colloid particles and promising research trends in basic research and potential applications were highlighted. Promising studies in this field include (1) clarifying bioavailability and evolution of activity of active ingredients in Pickering HIPEs by oral administration, (2) constructing a Pickering interfacial catalysis platform using protein colloidal particles, and (3) expanding the emerging applications of Pickering HIPEs in fields, such as partially hydrogenated oil replacers, probiotic encapsulation, and the template for porous materials.

Protein-polysaccharide complexes can be created in various ways (physical mixing, enzymatic cross-linking, chemical cross-linking, and Maillard reaction), and diverse protein-polysaccharide complexes are generally grouped into non-covalent and covalent complexes. Delivery systems constructed through assembly of protein-polysaccharide complexes (DSAPC) consist of emulsion-based delivery systems, capsule-based delivery systems, molecular complexes, nanogels, core-shell particles, composite nanoparticles, and micelles. DSAPC are effective delivery vehicles in enhancing the overall efficacy of bioactive ingredients, and DSAPC may possess multiple advantages over other delivery vehicles in bioactive ingredient delivery. However, designing and applying DSAPC are still faced with some challenges, such as low loading of bioactive ingredients. Efforts are required to reconsider and improve efficiency of DSAPC in many aspects, such as controlled release and targeted delivery. On the basis of more comprehensive and deeper understandings, DSAPC can be designed more rationally for delivery of bioactive ingredients.

Made available in DSpace on 2018-12-11T16:45:29Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-03-08 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) This paper describes the fabrication of 2D and 3D microfluidic paper-based analytical devices (μPADs) for monitoring glucose, total protein, and nitrite in blood serum and artificial urine. A new method of cutting and sealing filter paper to construct μPADs was demonstrated. Using an inexpensive home cutter printer soft cellulose-based filter paper was easily and precisely cut to produce pattern hydrophilic microchannels. 2D and 3D μPADs were designed with three detection zones each for the colorimetric detection of the analytes. A small volume of samples was added to the μPADs, which was photographed after 15 min using a digital camera. Both μPADs presented an excellent analytical performance for all analytes. The 2D device was applied in artificial urine samples and reached limits of detection (LODs) of 0.54 mM, 5.19 μM, and 2.34 μM for glucose, protein, and nitrite, respectively. The corresponding LODs of the 3D device applied for detecting the same analytes in artificial blood serum were 0.44 mM, 1.26 μM, and 4.35 μM. Departamento de Química Universidade Federal de São Carlos Intituto de Química-UNESP, Av. Francisco Degni s/n Intituto de Química-UNESP, Av. Francisco Degni s/n CNPq: 140484/2013-2 FAPESP: 2015/19890-1 CNPq: 427727/2016-2

Coomassie Brilliant Blue (CBB), used to stain protein gels, is known to be toxic. Therefore, laboratories do not discard used CBB into the sink owing to the possibility of it contaminating drinking water supplies. We tested the ability of various paper adsorbents to adsorb CBB released from gels during destaining. The efficiency was as follows-Kimwipes Teri towels multifold towels Whatman numbers 1 and 3 filter papers. Addition of three Kimwipes during destaining helped adsorb the dye released from a CBB-stained mini-gel. Stain removal with Kimwipes helps reduce destain use, organic waste accumulation, enable recycling of nonradioactive destaining solution and is 7.5-fold cheaper than an available method for CBB disposal. Next, we used Kimwipes to deplete the dye from a used CBB staining solution awaiting proper disposal by our Institutional Safety Office. Seventy five Kimwipes successfully helped remove the dye from a 0.05% CBB staining solution in 5-10 min. The blue Kimwipes did not release the CBB stain even when squeezed dry after incubation in various salts, water, or acid solutions for 5 weeks. The CBB removed thus can be simply disposed of as solid waste and will not leach out from solid landfills. Kimwipes, thus, enables CBB disposal in an environmentally friendly manner and allows for recycling of destaining solution.

Microfluidic paper-based analytical devices (μPADs) for molecular detection have great potential in the field of point-of-care diagnostics. Currently, a critical problem being faced by μPADs is improving their detection sensitivity. Various preconcentration processes have been developed, but they still have complicated structures and fabrication processes to integrate into μPADs. To address this issue, we have developed a novel paper-based preconcentrator utilizing ion concentration polarization (ICP) with minimal addition on lateral-flow paper. The cation selective membrane (i.e., Nafion) is patterned on adhesive tape, and this tape is then attached to paper-based channels. When an electric field is applied across the Nafion, ICP is initiated to preconcentrate the biomolecules in the paper channel. Departing from previous paper-based preconcentrators, we maintain steady lateral fluid flow with the separated Nafion layer; as a result, fluorescent dyes and proteins (FITC–albumin and bovine serum albumin) are continuously delivered to the preconcentration zone, achieving high preconcentration performance up to 1000-fold. In addition, we demonstrate that the Nafion-patterned tape can be integrated with various geometries (multiplexed preconcentrator) and platforms (string and polymer microfluidic channel). This work would facilitate integration of various ICP devices, including preconcentrators, pH/concentration modulators, and micro mixers, with steady lateral flows in paper-based platforms.

A novel integrated thermophilic submerged aerobic membrane bioreactor (TSAMBR) and electrochemical oxidation (EO) technology was developed for thermomechanical pulping pressate treatment with the aim of system closure. The TSAMBR was able to achieve a chemical oxygen demand (COD) removal efficiency of 88.6 ± 1.9-92.3 ± 0.7% under the organic loading rate of 2.76 ± 0.13-3.98 ± 0.23 kg COD/(m(3) d). An optimal hydraulic retention time (HRT) of 1.1 ± 0.1d was identified for COD removal. Cake formation was identified as the dominant mechanism of membrane fouling. The EO of the TSAMBR permeate was performed using a Ti/SnO(2)-Sb(2)O(5)-IrO(2) electrode. After 6-h EO, a complete decolourization was achieved and the COD removal efficiency was increased to 96.2 ± 1.2-98.2 ± 0.3%. The high-quality effluent produced by the TSAMBR-EO system can be reused as process water for system closure in pulp and paper mill.

An increasing number of publications include modeling. Often, such studies help us to gain a deeper insight into the phenomena studied and break down barriers between experimental and theoretical communities. However, combining experimental and theoretical work is challenging for authors, reviewers, and readers. To help maximize the usefulness and impact of combined theoretical and experimental research, this Primer describes the purpose, usefulness, and different types of models and addresses the practical aspect of integrated publications by outlining characteristics of good modeling, presentation, and fruitful collaborations.

This paper described a convenient semiquantitative method for colorimetric detection of protein with self-calibration integrated on the test strip. Hydrophilic paper was employed as microfluidic device for running colorimetric assay, tree-shaped design was developed to ensure uniform microfluidic flow for multiple branches. The approach was validated with bovine serum albumin (BSA) colorimetric detection, and colorimetric results observed by naked eyes were consistent with that from apparatus. The device could be coupled with digital transmission of images for remote monitoring system for diagnosis, food control, and environmental analysis.

Paper has been proposed as an inexpensive and versatile carrier for microfluidics devices with abilities well beyond simple capillary action for pregnancy tests and the like. Unlike standard microfluidics devices, extracting a fluid from the paper is a challenge and a drawback to its broader use. Here, we extract fluid from narrow paper strips using surface acoustic wave (SAW) irradiation that subsequently atomizes the extracted fluid into a monodisperse aerosol for use in mass spectroscopy, medical diagnostics, and drug delivery applications. Two protein molecules, ovalbumin and bovine serum albumin (BSA), have been preserved in paper and then extracted using atomized mist through SAW excitation; protein electrophoresis shows there is less than 1% degradation of either protein molecule in this process. Finally, a solution of live yeast cells was infused into paper, which was subsequently dried for preservation then remoistened to extract the cells via SAW atomization, yielding live cells at the completion of the process. The successful preservation and extraction of fluids, proteins and yeast cells significantly expands the usefulness of paper in microfluidics.

Two autophagy-related (ATG) ubiquitin-like conjugation systems, the ATG12 and ATG8 systems, play important roles in macroautophagy. While multiple duplications and losses of the ATG conjugation system proteins are found in different lineages, the extent to which the underlying systems diversified across eukaryotes is not fully understood. Here, in order to understand the evolution of the ATG conjugation systems, we constructed a transcriptome database consisting of 94 eukaryotic species covering major eukaryotic clades and systematically identified ATG conjugation system components. Both ATG10 and the C-terminal glycine of ATG12 are essential for the canonical ubiquitin-like conjugation of ATG12 and ATG5. However, loss of ATG10 or the C-terminal glycine of ATG12 occurred at least 16 times in a wide range of lineages, suggesting that possible covalent-to-non-covalent transition is not limited to the species that we previously reported such as Alveolata and some yeast species. Some species have only the ATG8 system (with conjugation enzymes) or only ATG8 (without conjugation enzymes). More than 10 species have ATG8 homologs without the conserved C-terminal glycine, and Tetrahymena has an ATG8 homolog with a predicted transmembrane domain, which may be able to anchor to the membrane independent of the ATG conjugation systems. We discuss the possibility that the ancestor of the ATG12 and ATG8 systems is more similar to ATG8. Overall, our study offers a whole picture of the evolution and diversity of the ATG conjugation systems among eukaryotes, and provides evidence that functional diversifications of the systems are more common than previously thought. Abbreviations: APEAR: ATG8–PE association region; ATG: autophagy-related; LIR: LC3-interacting region; NEDD8: neural precursor cell expressed, developmentally down-regulated gene 8; PE: phosphatidylethanolamine; SAMP: small archaeal modifier protein; SAR: Stramenopiles, Alveolata, and Rhizaria; SMC: structural maintenance of chromosomes; SUMO: small ubiquitin like modifier; TACK: Thaumarchaeota, Aigarchaeota, Crenarchaeota, and Korarchaeota; UBA: ubiquitin like modifier activating enzyme; UFM: ubiquitin fold modifier; URM: ubiquitin related modifier.

Aquatic habitat is affected by paper mill effluent discharge in many ways. The effect of paper mill effluent on the gill ATPases was studied in freshwater fish Channa punctata (Bloch) exposed to 1%(v/v) of effluent for 15, 30, and 60 days. There was a time-dependent significant (P0.05-0.001) decrease in all the ATPase activities measured, viz., total, Na(+), K(+)--and ouabain-insensitive ATPase in gill. ATPases play an important role in maintenance of functional integrity of plasma membrane and in several intracellular functions and are considered to be a sensitive indicator of toxicity. In addition to this, branchial ATPases are intimately involved in osmoregulation, acid-base regulation, and respiration of fish. The inhibition of ATPases in gills by, e.g., paper mill effluent could cause disruption of these processes. It is suggested that measurement of ATPases could also be used as a surrogate biomarker of exposure to chemical pollutants.

We present an origami paper-based electrophoretic device (oPAD-Ep) that achieves rapid (∼5 min) separation of fluorescent molecules and proteins. Due to the innovative design, the required driving voltage is just ∼10 V, which is more than 10 times lower than that used for conventional electrophoresis. The oPAD-Ep uses multiple, thin (180 μm/layer) folded paper layers as the supporting medium for electrophoresis. This approach significantly shortens the distance between the anode and cathode, and this, in turn, accounts for the high electric field (>1 kV/m) that can be achieved even with a low applied voltage. The multilayer design of the oPAD-Ep enables convenient sample introduction by use of a slip layer as well as easy product analysis and reclamation after electrophoresis by unfolding the origami paper and cutting out desired layers. We demonstrate the use of oPAD-Ep for simple separation of proteins in bovine serum, which illustrates its potential applications for point-of-care diagnostic testing.

Conventional microfluidic devices are typically complex and expensive. The devices require the use of pneumatic control systems or highly precise pumps to control the flow in the devices. This work investigates an alternative method using paper based microfluidic devices to replace conventional microfluidic devices. Size based separation and extraction experiments conducted were able to separate free dye from a mixed protein and dye solution. Experimental results showed that pure fluorescein isothiocyanate could be separated from a solution of mixed fluorescein isothiocyanate and fluorescein isothiocyanate labeled bovine serum albumin. The analysis readings obtained from a spectrophotometer clearly show that the extracted tartrazine sample did not contain any amount of Blue-BSA, because its absorbance value was 0.000 measured at a wavelength of 590nm, which correlated to Blue-BSA. These demonstrate that paper based microfluidic devices, which are inexpensive and easy to implement, can potentially replace their conventional counterparts by the use of simple geometry designs and the capillary action. These findings will potentially help in future developments of paper based microfluidic devices.

The passivation of nonspecific protein adsorption to paper is a major barrier to the use of paper as a platform for microfluidic bioassays. Herein we describe a simple, scalable protocol based on adsorption and cross-linking of poly(oligoethylene glycol methacrylate) (POEGMA) derivatives that reduces nonspecific adsorption of a range of proteins to filter paper by at least 1 order of magnitude without significantly changing the fiber morphology or paper macroporosity. A lateral-flow test strip coated with POEGMA facilitates effective protein transport while also confining the colorimetric reporting signal for easier detection, giving improved performance relative to bovine serum albumin (BSA)-blocked paper. Enzyme-linked immunosorbent assays based on POEGMA-coated paper also achieve lower blank values, higher sensitivities, and lower detection limits relative to ones based on paper blocked with BSA or skim milk. We anticipate that POEGMA-coated paper can function as a platform for the design of portable, disposable, and low-cost paper-based biosensors.

Type 1 diabetes (T1D) is increasing in incidence and predictable with measurement of serum islet autoantibodies (iAb) years prior to clinical disease onset. Identifying iAb positive individuals reduces diabetic ketoacidosis and identifies individuals for T1D prevention trials. However, large scale screening for iAb remains challenging as assays have varying sensitivities and specificities, insulin autoantibodies remain difficult to measure and venipuncture is generally required to obtain serum. We developed an approach to reliably measure all four major iAb, including insulin autoantibodies, from dried blood spots (DBS) on filter-paper. By spiking iAb positive serum into iAb negative whole blood in a dose titration, we optimized the conditions for autoantibody elution from filter paper as measured by fluid phase radioimmunoassays. After assessing stability of measuring iAb from DBS over time, we then screened iAb from DBS and the corresponding serum in new-onset T1D (n = 52), and controls (n = 72) which included first-degree relatives of T1D patients. iAb measured from eluted DBS in new-onset T1D strongly correlated with serum measurements (R2 = 0.96 for mIAA, GADA = 0.94, IA-2A = 0.85, ZnT8A = 0.82, p

Interest in applications of protein crystallography to medicine was evident, as the first high-resolution structures emerged in the 50s and 60s. In Cambridge, Max Perutz and John Kendrew sought to understand mutations in sickle cell and other genetic diseases related to hemoglobin, while in Oxford, the group of Dorothy Hodgkin became interested in long-lasting zinc-insulin crystals for treatment of diabetes and later considered insulin redesign, as synthetic insulins became possible. The use of protein crystallography in structure-guided drug discovery emerged as enzyme structures allowed the identification of potential inhibitor-binding sites and optimization of interactions of hits using the structure of the target protein. Early examples of this approach were the use of the structure of renin to design antihypertensives and the structure of HIV protease in design of AIDS antivirals. More recently, use of structure-guided design with fragment-based drug discovery, which reduces the size of screening libraries by decreasing complexity, has improved ligand efficiency in drug design and has been used to progress three oncology drugs through clinical trials to FDA approval. We exemplify current developments in structure-guided target identification and fragment-based lead discovery with efforts to develop new antimicrobials for mycobacterial infections.

Bladder cancer remains one of the most common forms of cancer and yet there are limited small molecule targeted therapies. Here, we present a computational platform to identify new potential targets for bladder cancer therapy. Our method initially exploited a set of known driver genes for bladder cancer combined with predicted bladder cancer genes from mutationally enriched protein domain families. We enriched this initial set of genes using protein network data to identify a comprehensive set of 323 putative bladder cancer targets. Pathway and cancer hallmarks analyses highlighted putative mechanisms in agreement with those previously reported for this cancer and revealed protein network modules highly enriched in potential drivers likely to be good targets for targeted therapies. 21 of our potential drug targets are targeted by FDA approved drugs for other diseases — some of them are known drivers or are already being targeted for bladder cancer (FGFR3, ERBB3, HDAC3, EGFR). A further 4 potential drug targets were identified by inheriting drug mappings across our in-house CATH domain functional families (FunFams). Our FunFam data also allowed us to identify drug targets in families that are less prone to side effects i.e., where structurally similar protein domain relatives are less dispersed across the human protein network. We provide information on our novel potential cancer driver genes, together with information on pathways, network modules and hallmarks associated with the predicted and known bladder cancer drivers and we highlight those drivers we predict to be likely drug targets.

In this report, we describe a simple, low-cost, straight forward and highly reproducible fabrication method of microfluidic systems. This system was cut on a glass fiber membrane by a common cutter without using any other sophisticated equipment or organic solvents. This format represents a novel type of paper-based microfluidics with high resolution of the microchannel down to ~137 μm, comparable to those made by conventional photolithography. We successfully applied this method to microfluidics to create a star micro-array format of multiplexed urine tests in this study.