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1.
ACS Omega ; 6(34): 22439-22446, 2021 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-34497933

RESUMO

Colorimetric assays typically offer a rapid and convenient method to assess analytes that span healthcare monitoring to water quality testing. However, such tests can only provide qualitative results when employed in resource-limited settings or require bulky and expensive equipment such as lab spectrophotometers to allow quantitative measurements. In this paper, we report on the use of a handheld colorimeter to quantitatively determine the concentration of analytes in a manner that is independent of ambient lighting or initial sample color. The method combines the response of the sensor with first-principles modeling that better describes the nature of the assay compared to linear-in-parameters regression modeling that is typically performed in other studies. This method was successfully demonstrated using a number of colorimetric assays: (1) determination of solution pH using a universal indicator, (2) quantification of the DNase presence using a DNA-gold nanoparticle assay, and (3) quantification of the concentration of the antibiotic tetracycline using a cell-based assay.

2.
ACS Nano ; 15(9): 13943-13969, 2021 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-34524790

RESUMO

Since their discovery almost three decades ago, DNAzymes have been used extensively in biosensing. Depending on the type of DNAzyme being used, these functional oligonucleotides can act as molecular recognition elements within biosensors, offering high specificity to their target analyte, or as reporters capable of transducing a detectable signal. Several parameters need to be considered when designing a DNAzyme-based biosensor. In particular, given that many of these biosensors immobilize DNAzymes onto a sensing surface, selecting an appropriate immobilization strategy is vital. Suboptimal immobilization can result in both DNAzyme detachment and poor accessibility toward the target, leading to low sensing accuracy and sensitivity. Various approaches have been employed for DNAzyme immobilization within biosensors, ranging from amine and thiol-based covalent attachment to non-covalent strategies involving biotin-streptavidin interactions, DNA hybridization, electrostatic interactions, and physical entrapment. While the properties of each strategy inform its applicability within a proposed sensor, the selection of an appropriate strategy is largely dependent on the desired application. This is especially true given the diverse use of DNAzyme-based biosensors for the detection of pathogens, metal ions, and clinical biomarkers. In an effort to make the development of such sensors easier to navigate, this paper provides a comprehensive review of existing immobilization strategies, with a focus on their respective advantages, drawbacks, and optimal conditions for use. Next, common applications of existing DNAzyme-based biosensors are discussed. Last, emerging and future trends in the development of DNAzyme-based biosensors are discussed, and gaps in existing research worthy of exploration are identified.


Assuntos
Técnicas Biossensoriais , DNA Catalítico
3.
Acc Chem Res ; 54(18): 3540-3549, 2021 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-34478272

RESUMO

Pathogens have long presented a significant threat to human lives, and hence the rapid detection of infectious pathogens is vital for improving human health. Current detection methods lack the means to detect infectious pathogens in a simple, rapid, and reliable manner at the time and point of need. Functional nucleic acids (FNAs) have the potential to overcome these limitations by acting as key components for point-of-care (POC) biosensors due to their distinctive advantages that include high binding affinities and specificities, excellent chemical stability, ease of synthesis and modification, and compatibility with a variety of signal-amplification and signal-transduction mechanisms.This Account summarizes the work completed in our groups toward developing FNA-based biosensors for detecting bacteria. In vitro selection has led to the isolation of many RNA-cleaving fluorogenic DNAzymes (RFDs) and DNA aptamers that can recognize infectious pathogens, including Escherichia coli, Clostridium difficile, Helicobacter pylori, and Legionella pneumophila. In most cases, a "many-against-many" approach was employed using a DNA library against a crude cellular mixture of an infectious pathogen containing diverse biomarkers as the target to isolate RFDs, with combined counter and positive selections ensuring high specificity toward the desired target. This procedure allows for the isolation of pathogen-specific FNAs without first identifying a suitable biomarker. Multiple target-specific DNA aptamers, including anti-glutamate dehydrogenase (GDH) circular aptamers, anti-degraded toxin B aptamers, and anti-RNase HII aptamers, have also been isolated for the detection of bacteria such as Clostridium difficile. The isolated FNAs have been integrated into fluorescent, colorimetric, and electrochemical biosensors using various signal transduction mechanisms. Both simple-to-use paper-based analytical devices and hand-held electrical devices with integrated FNAs have been developed for POC applications. In addition, signal-amplification strategies, including DNA catenane enabled rolling circle amplification (RCA), DNAzyme feedback RCA, and an all-DNA amplification system using a four-way junction and catalytic hairpin assembly (CHA), have been designed and applied to these systems to further increase their detection sensitivity. The use of these FNA-based biosensors to detect pathogens directly in clinical samples, such as urine, blood, and stool, has now been demonstrated with an outstanding sensitivity of as low as 10 cells per milliliter, highlighting the tremendous potential of using FNA-based sensors in clinical applications. We further describe strategies to overcome the challenges of using FNA-based biosensors in clinical applications, including strategies to improve the stability of FNAs in biological samples and prevent their nonspecific degradation from nucleases and strategies to deal with issues such as signal loss caused by nonspecific binding and biofouling. Finally, the remaining roadblocks for employing FNA-based biosensors in clinical applications are discussed.


Assuntos
Aptâmeros de Nucleotídeos/metabolismo , Bactérias/genética , Técnicas Biossensoriais/métodos , DNA Catalítico/metabolismo , Aptâmeros de Nucleotídeos/química , Bactérias/isolamento & purificação , DNA Catalítico/química , Escherichia coli/genética , Escherichia coli/isolamento & purificação , Técnicas de Amplificação de Ácido Nucleico , Sistemas Automatizados de Assistência Junto ao Leito
4.
Angew Chem Int Ed Engl ; 60(45): 24266-24274, 2021 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-34464491

RESUMO

We report a simple and rapid saliva-based SARS-CoV-2 antigen test that utilizes a newly developed dimeric DNA aptamer, denoted as DSA1N5, that specifically recognizes the spike proteins of the wildtype virus and its Alpha and Delta variants with dissociation constants of 120, 290 and 480 pM, respectively, and binds pseudotyped lentiviruses expressing the wildtype and alpha trimeric spike proteins with affinity constants of 2.1 pM and 2.3 pM, respectively. To develop a highly sensitive test, DSA1N5 was immobilized onto gold electrodes to produce an electrochemical impedance sensor, which was capable of detecting 1000 viral particles per mL in 1:1 diluted saliva in under 10 min without any further sample processing. Evaluation of 36 positive and 37 negative patient saliva samples produced a clinical sensitivity of 80.5 % and specificity of 100 % and the sensor could detect the wildtype virus as well as the Alpha and Delta variants in the patient samples, which is the first reported rapid test that can detect any emerging variant of SARS-CoV-2.


Assuntos
Antígenos Virais/análise , Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais , Teste Sorológico para COVID-19 , Técnicas Eletroquímicas , SARS-CoV-2/genética , Humanos , Saliva/química
5.
Nucleic Acids Res ; 49(13): 7267-7279, 2021 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-34232998

RESUMO

We performed in vitro selection experiments to identify DNA aptamers for the S1 subunit of the SARS-CoV-2 spike protein (S1 protein). Using a pool of pre-structured random DNA sequences, we obtained over 100 candidate aptamers after 13 cycles of enrichment under progressively more stringent selection pressure. The top 10 sequences all exhibited strong binding to the S1 protein. Two aptamers, named MSA1 (Kd = 1.8 nM) and MSA5 (Kd = 2.7 nM), were assessed for binding to the heat-treated S1 protein, untreated S1 protein spiked into 50% human saliva and the trimeric spike protein of both the wildtype and the B.1.1.7 variant, demonstrating comparable affinities in all cases. MSA1 and MSA5 also recognized the pseudotyped lentivirus of SARS-CoV-2 with respective Kd values of 22.7 pM and 11.8 pM. Secondary structure prediction and sequence truncation experiments revealed that both MSA1 and MSA5 adopted a hairpin structure, which was the motif pre-designed into the original library. A colorimetric sandwich assay was developed using MSA1 as both the recognition element and detection element, which was capable of detecting the pseudotyped lentivirus in 50% saliva with a limit of detection of 400 fM, confirming the potential of these aptamers as diagnostic tools for COVID-19 detection.


Assuntos
Aptâmeros de Nucleotídeos , COVID-19/virologia , Biblioteca Gênica , Mutação , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genética , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/genética , Pareamento de Bases , Sequência de Bases , COVID-19/diagnóstico , Colorimetria/métodos , Humanos , Conformação de Ácido Nucleico , Técnica de Seleção de Aptâmeros
6.
Angew Chem Int Ed Engl ; 60(9): 4782-4788, 2021 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-33188548

RESUMO

Legionella pneumophila is a deadly bacterial pathogen that has caused numerous Legionnaires' disease outbreaks, where cooling towers were the most common source of exposure. Bacterial culturing is used for L. pneumophila detection, but this method takes approximately 10 days to complete. In this work, an RNA-cleaving fluorogenic DNAzyme, named LP1, was isolated. Extensive characterization revealed that LP1 is reactive with multiple infectious isolates of L. pneumophila but inactive with 25 other common bacterial species. LP1 is likely activated by a protein target, capable of generating a detectable signal in the presence of as few as 10 colony-forming units of L. pneumophila, and able to maintain its activity in cooling tower water from diverse sources. Given that similar DNAzymes have been incorporated into many sensitive assays for bacterial detection, LP1 holds the potential for the development of biosensors for monitoring the contamination of L. pneumophila in exposure sources.


Assuntos
DNA Catalítico/metabolismo , Legionella pneumophila/genética , RNA/metabolismo , Técnicas Biossensoriais , DNA Catalítico/química , DNA Catalítico/isolamento & purificação , Cinética , Conformação de Ácido Nucleico , Clivagem do RNA , Microbiologia da Água
7.
Chembiochem ; 21(5): 632-637, 2020 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-31544309

RESUMO

The engineering of easy-to-use biosensors with ultra-low detection sensitivity remains a major challenge. Herein, we report a simple approach for creating such sensors through the use of an RNA-cleaving DNAzyme (RcD) and a strategy designed to concentrate its cleavage product significantly. The assay uses micron-sized beads loaded with a target-responsive RcD and a paper strip containing a microzone covered with a DNA oligonucleotide capable of capturing the cleavage product of the RcD through Watson-Crick hybridization. Placing the beads and the paper strip in a target-containing test sample allows the bead-bound RcD molecules to undergo target-induced RNA cleavage, releasing a DNA fragment that is captured by the paper strip. This strategy, though simple, is very effective in achieving high levels of detection sensitivity, being able to enrich the concentration of the cleavage product by three orders of magnitude. It is also compatible with both fluorescence-based and colorimetric reporting mechanisms. This work provides a simple platform for developing ultrasensitive biosensors that take advantage of the widely available RcDs as molecular recognition elements.


Assuntos
Técnicas Biossensoriais , DNA Catalítico/química , Nanotecnologia , Oligodesoxirribonucleotídeos/química , RNA/química , Escherichia coli , Hibridização de Ácido Nucleico , Clivagem do RNA
8.
Environ Sci Technol ; 54(1): 184-194, 2020 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-31790215

RESUMO

As a proof of concept, a rapid assay consisting of a cell-based biosensor (CBB) panel of pure bacterial strains, a fluorescent dye, and partial least squares (PLS) modeling was developed to assess the nitrification inhibition potential of industrial wastewater (WW) samples. The current standard method used to assess the nitrification inhibition potential is the specific nitrification rate (SNR) batch test, which requires approximately 4 h to complete under the watch of an experienced operator. In this study, we exposed the CBB panel of seven bacterial strains (nitrifying and non-nitrifying) to 28 different industrial WW samples and then probed both the membrane integrity and cellular activity using a commercially available "live/dead" fluorescent dye. The CBB panel response acts as a surrogate measurement for the performance of nitrification. Of the seven strains, four (Nitrospira, Escherichia coli, Bacillus subtilis, Bacillus cereus) were identified via the modeling technique to be the most significant contributors for predicting the nitrification inhibition potential. The key outcome from this work is that the CBB panel fluorescence data (collected in approximately 10 min) can accurately predict the outcome of an SNR batch test (that takes 4 h) when performed with the same WW samples and has a strong potential to approximate the chemical composition of these WW samples using PLS modeling. Overall, this is a powerful technique that can be used for point-of-use detection of nitrification inhibition.


Assuntos
Reatores Biológicos , Nitrificação , Amônia , Bactérias , Análise dos Mínimos Quadrados , Nitritos , Águas Residuárias
9.
Angew Chem Int Ed Engl ; 58(29): 9907-9911, 2019 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-31095864

RESUMO

The reliable detection of pathogenic bacteria in complex biological samples using simple assays or devices remains a major challenge. Herein, we report a simple colorimetric paper device capable of providing specific and sensitive detection of Helicobacter pylori (H. pylori), a pathogen strongly linked to gastric carcinoma, gastric ulcers, and duodenal ulcers, in stool samples. The sensor molecule, an RNA-cleaving DNAzyme obtained through in vitro selection, is activated by a protein biomarker from H. pylori. The colorimetric paper sensor, designed on the basis of the RNA-cleaving property of the DNAzyme, is capable of sensitive detection of H. pylori in human stool samples with minimal sample processing and provides results in minutes. It remains fully functional under storage at ambient temperature for at least 130 days. This work lays a foundation for developing DNAzyme-enabled paper-based point-of-care diagnostic devices for monitoring pathogens in complex samples.


Assuntos
Técnicas Biossensoriais/métodos , Colorimetria/métodos , DNA Catalítico/metabolismo , Infecções por Helicobacter/diagnóstico , Helicobacter pylori/patogenicidade , Humanos
10.
Sci Rep ; 9(1): 7631, 2019 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-31113974

RESUMO

Most currently available vaccines, particularly live vaccines, require the cold chain, as vaccine efficacy can be significantly hampered if they are not stored in a temperature range of 2-8 °C at all times. This necessity places a tremendous financial and logistical burden on vaccination programs, particularly in the developing world. The development of thermally stable vaccines can greatly alleviate this problem and, in turn, increase vaccine accessibility worldwide. In this paper, we detail a simple and cost-effective method for stabilizing live vaccines that uses FDA-approved materials. To this end, we dried enveloped DNA (Herpes Simplex Virus type 2) and RNA (Influenza A virus) viral vaccines in a pullulan and trehalose mixture. The results of these studies showed that the live-attenuated HSV-2 vaccine retained its efficacy for at least 2 months of storage at 40 °C, while the inactivated influenza vaccine was able to retain its immunogenicity for at least 3 months of storage at 40 °C. This work presents a simple approach that allows thermo-sensitive vaccines to be converted into thermo-stable vaccines that do not require refrigeration, thus contributing to the improvement of vaccine deployment throughout the world.


Assuntos
Vacinas contra o Vírus do Herpes Simples/química , Ácidos Nucleicos Imobilizados/química , Vacinas contra Influenza/química , Membranas Artificiais , Potência de Vacina , Animais , Chlorocebus aethiops , Custos e Análise de Custo , DNA Viral/química , DNA Viral/imunologia , Cães , Vacinas contra o Vírus do Herpes Simples/economia , Vacinas contra o Vírus do Herpes Simples/imunologia , Ácidos Nucleicos Imobilizados/imunologia , Imunogenicidade da Vacina , Vacinas contra Influenza/economia , Vacinas contra Influenza/imunologia , Células Madin Darby de Rim Canino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , RNA Viral/química , RNA Viral/imunologia , Açúcares/química , Células Vero
11.
Langmuir ; 35(16): 5517-5524, 2019 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-30924655

RESUMO

Piezoelectric inkjet printing is susceptible to printhead clogging when printing with inks that contain dispersed particles. This paper investigates the mechanisms by which 28-530 nm nanoparticle dispersions induce printhead clogging without forming large aggregates or thick deposited layers on printhead surfaces. Printing experiments were combined with nanoparticle deposition studies and with experiments where inks were pumped through printheads at a constant flow rate with a syringe pump. Submonolayer coverages of hydrophobic cationic polystyrene nanoparticles adhering to printhead surfaces promote rapid clogging by trapped air that enters from the nozzle opening. We propose that the deposited particles distort the shape of the ink/air meniscus, possibly causing air entrainment, and promote air bubble adhesion to the interior printhead surfaces. The printer's purge-blot cleaning procedure removes air clogs, but the clogs quickly reform when printing is resumed because the adsorbed nanoparticles are not removed by the cleaning procedure. Nondepositing anionic hydrophobic nanoparticles cause much less clogging, possibly because of filtration of trace large aggregates. Colloidal stability is a necessary but not sufficient criterion for ink dispersions; the ink particles must not adsorb onto the printhead surfaces. Thus, alternate surface chemistries for the printhead and ink particle surfaces may be required to print hydrophobic ink materials.

12.
ACS Appl Mater Interfaces ; 11(4): 4318-4327, 2019 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-30615426

RESUMO

Highly sensitive pressure sensors are usually made from soft materials that allow large deformations to be obtained when very small pressures are applied. Unfortunately, this current paradigm limits the ability to create sensors capable of high sensitivities and broad dynamic ranges as these materials are prone to saturation responses when attempting to obtain measurements involving high pressures. In this paper, we detail a piezoresistive pressure sensor that is capable of high sensitivity over a pressure range spanning from 0.6 Pa (a mosquito touching a surface) to 200 kPa (an elephant standing on the surface). The sensor's ability to cover such a broad dynamic range is made possible by the fairly hard foam used in its construction as this material is capable of propagating strain in a highly effective manner due to its hierarchical porous structure. The material was fabricated by using high-internal-phase emulsion (HIPE) as a template to generate a highly porous material consisting of small pores packed between larger ones whose inner walls are lined with reduced graphene oxide. The developed foam exhibits very fast response times (less than 15.4 ms) and excellent cyclic stability (at least 10,000 cycles). Furthermore, it is capable of responding to the entire tactile pressure range, and it can be formatted as pixelated arrays, which makes it highly suitable for integration into wearable electronic devices. Such arrays were built and used to identify and render the shape of objects with different geometries, including a sphere, a triangle, a square, and two nearly identical rods differing only by 0.4 mm in diameter.

13.
Chemosphere ; 221: 45-54, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30634148

RESUMO

Biocides, also referred to as 'microbicides' or 'inhibitors', are widely used in industrial processes (e.g. utility water in cooling towers) to control and/or eliminate the growth of microorganisms. Because of their inherent toxicity, their presence in various sources (e.g. river sediments, potable water) can negatively affect ecosystems. Currently available biocide detection techniques are not suitable for 'point-of-use' applications since they are tedious, complicated, and often require experienced personnel to operate. To address this concern, we sought to develop a simple-to-use toxicity bioassay based on a model microorganism (E. coli) after short (<30 min) exposure to known biocides that can be stored at room temperature (preferably) or in the fridge. Based on recent work and our expertise in polymer-based preservation of biomolecules, we leveraged this knowledge to improve E. coli preservation for biocide detection purposes. A design-of-experiments strategy was used to evaluate 16 different preservation conditions from 5 process parameters (i.e. 25-1 fractional factorial). It was found that pullulan, a sugar-based polymer, improved E. coli culturability by an order of magnitude after three months of storage. Also, it was found that storing E. coli in the fridge in Milli-Q water was favorable for maintaining a high level of culturability. Finally, the toxicity of three common biocides (Cetyltrimethylammonium bromide (CTAB), ProClin™ 300, and Grotan® BK) was evaluated using a fluorescence-based assay across all 16 preservation conditions. The response of the preserved E. coli was biocide specific and at certain conditions did not vary during the entire three-month storage period.


Assuntos
Desinfetantes/análise , Preservação Biológica/métodos , Testes de Toxicidade Aguda/métodos , Bactérias/efeitos dos fármacos , Bioensaio/métodos , Desinfetantes/farmacologia , Escherichia coli/efeitos dos fármacos
14.
Biotechnol Prog ; 35(2): e2764, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30536873

RESUMO

Rolling circle amplification (RCA), polymerase chain reaction (PCR), and loop-mediated isothermal amplification (LAMP), are powerful tools that can be used for gene manipulation, pathogen detection, and infectious disease diagnostics. However, these techniques require trained personnel, as the pipetting steps involved can lead to contamination and, consequently, erroneous results. Furthermore, many of the reagents used in molecular biology are thermally labile and must be kept within a cold-chain. In this article, we present a simple and cost-effective method that allows molecular biology reagents to be thermally stabilized into ready-to-use mastermixes via drying in pullulan and trehalose films. Our experimental results demonstrate that this method is capable of preserving the activity of RCA, PCR, LAMP, ligase, polynucleotide kinase, and Klenow fragment mastermixes for at least 3 months at ambient conditions. Thus, stabilizing reagents via drying in pullulan and trehalose film may allow for a drastic reduction in the number of pipetting steps and the elimination of the need for a cold chain. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2764, 2019.


Assuntos
Ligases/metabolismo , Biologia Molecular , Polinucleotídeo 5'-Hidroxiquinase/metabolismo , Temperatura , Estabilidade Enzimática , Técnicas de Amplificação de Ácido Nucleico , Reação em Cadeia da Polimerase
15.
ACS Nano ; 12(4): 3287-3294, 2018 04 24.
Artigo em Inglês | MEDLINE | ID: mdl-29621883

RESUMO

Here, we report the development of a transparent, durable, and flexible sensing surface that generates a fluorescence signal in the presence of a specific target bacterium. This material can be used in packaging, and it is capable of monitoring microbial contamination in various types of food products in real time without having to remove the sample or the sensor from the package. The sensor was fabricated by covalently attaching picoliter-sized microarrays of an E. coli-specific RNA-cleaving fluorogenic DNAzyme probe (RFD-EC1) to a thin, flexible, and transparent cyclo-olefin polymer (COP) film. Our experimental results demonstrate that the developed (RFD-EC1)-COP surface is specific, stable for at least 14 days under various pH conditions (pH 3-9), and can detect E. coli in meat and apple juice at concentrations as low as 103 CFU/mL. Furthermore, we demonstrate that our sensor is capable of detecting bacteria while still attached to the food package, which eliminates the need to manipulate the sample. The developed biosensors are stable for at least the shelf life of perishable packaged food products and provide a packaging solution for real-time monitoring of pathogens. These sensors hold the potential to make a significant contribution to the ongoing efforts to mitigate the negative public-health-related impacts of food-borne illnesses.


Assuntos
DNA Catalítico/química , Contaminação de Alimentos/análise , Embalagem de Alimentos , Sondas Moleculares/química , Impressão Tridimensional , Técnicas Biossensoriais , DNA Catalítico/metabolismo , Escherichia coli/isolamento & purificação , Fluorescência , Concentração de Íons de Hidrogênio , Sondas Moleculares/metabolismo , Polímeros/química , Polímeros/metabolismo , Propriedades de Superfície , Fatores de Tempo
16.
Anal Bioanal Chem ; 410(4): 1217-1230, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-28940009

RESUMO

Foodborne pathogens are a burden to the economy and a constant threat to public health. The ability to rapidly detect the presence of foodborne pathogens is a vital component of any strategy towards establishing a safe and secure food supply chain. Bacteriophages (phages) are viruses capable of infecting and replicating within bacteria in a strain-specific manner. The ubiquitous and selective nature of phages makes them ideal for the detection and biocontrol of bacteria. Therefore, the objective of this research was to develop and test a phage-based paper dipstick biosensor for the detection of various foodborne pathogens in food matrices. The first step was to identify the best method for immobilizing phages on paper such that their biological activity (infectivity) was preserved. It was found that piezoelectric inkjet printing resulted in lower loss of phage infectivity when compared with other printing methods (namely gravure and blade coating) and that ColorLok paper was ideally suited to create functional sensors. The phage-based bioactive papers developed with use of piezoelectric inkjet printing actively lysed their target bacteria and retained this antibacterial activity for up to 1 week when stored at room temperature and 80% relative humidity. These bioactive paper strips in combination with quantitative real-time PCR were used for quantitative determination of target bacteria in broth and food matrices. A phage dipstick was used to capture and infect Escherichia coli O157:H7, E. coli O45:H2, and Salmonella Newport in spinach, ground beef and chicken homogenates, respectively, and quantitative real-time PCR was used to detect the progeny phages. A detection limit of 10-50 colony-forming units per millilitre was demonstrated with a total assay time of 8 h, which was the duration of a typical work shift in an industrial setting. This detection method is rapid and cost-effective, and may potentially be applied to a broad range of bacterial foodborne pathogens. Graphical abstract ᅟ.


Assuntos
Colífagos , Microbiologia de Alimentos , Técnicas Biossensoriais , Contagem de Colônia Microbiana , Meios de Cultura , Escherichia coli O157/isolamento & purificação , Escherichia coli O157/patogenicidade , Limite de Detecção , Papel
17.
Biomacromolecules ; 19(1): 62-70, 2018 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-29168379

RESUMO

We report a simple method of preparing autonomous and rapid self-adhesive hydrogels and their use as building blocks for additive manufacturing of functional tissue scaffolds. Dynamic cross-linking between 2-aminophenylboronic acid-functionalized hyaluronic acid and poly(vinyl alcohol) yields hydrogels that recover their mechanical integrity within 1 min after cutting or shear under both neutral and acidic pH conditions. Incorporation of this hydrogel in an interpenetrating calcium-alginate network results in an interfacially stiffer but still rapidly self-adhesive hydrogel that can be assembled into hollow perfusion channels by simple contact additive manufacturing within minutes. Such channels withstand fluid perfusion while retaining their dimensions and support endothelial cell growth and proliferation, providing a simple and modular route to produce customized cell scaffolds.


Assuntos
Adesivos/química , Hidrogéis/química , Engenharia Tecidual/métodos , Tecidos Suporte , Ácidos Borônicos/química , Reagentes para Ligações Cruzadas/química , Células Epiteliais/citologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Ácido Hialurônico/química , Concentração de Íons de Hidrogênio , Álcool de Polivinil/química
18.
ACS Biomater Sci Eng ; 4(11): 3802-3808, 2018 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-33429601

RESUMO

The antimicrobial activity of LISTEX P100, Salmonella CG4, and E. coli AG10 bacteriophages were preserved in pullulan-trehalose mixture as dried films and as coatings on food packaging. The phages encapsulated in pullulan-trehalose films were able to retain infectivity for up to 3 months at ambient storage conditions. Various buffers, disaccharides and disaccharide concentrations were investigated to optimize the long-term stability of the phages in the films. It was found that pullulan and trehalose need to be simultaneously present in the film to provide the stabilizing effect and that the presence of buffers that lead to the formation of crystals in the films must be avoided for phage activity to be maintained. Overall, this study describes a method of preserving bacteriophage activity in a dried format that has great potential for use as coatings, which can be used to create antimicrobial surfaces for food preparation and for food preservation.

19.
Sci Rep ; 7(1): 12335, 2017 09 26.
Artigo em Inglês | MEDLINE | ID: mdl-28951563

RESUMO

We present a simple all-in-one paper-based sensor for E. coli detection using a composite ink made of a fluorogenic DNAzyme probe for bacterial recognition and signal generation, lysozyme that lyses whole bacterial cells, and pullulan/trehalose sugars that stabilize printed bioactive molecules. The paper sensor is capable of producing a fluorescence signal as a readout within 5 minutes upon contacting E. coli, can achieve a limit of detection of 100 cells/mL, in a variety of sample matrixes, without sample enrichment, and remains stable for at least 6 months when stored at ambient temperature. Therefore, this simple paper sensor provides rapid bacterial testing on site, and can be shipped and stored under ambient conditions to benefit users living in resource-limited regions.


Assuntos
Bactérias/isolamento & purificação , Técnicas Biossensoriais/métodos , Biotecnologia/métodos , Sondas Moleculares/química , Papel , Técnicas Biossensoriais/economia , DNA Catalítico/química , Enzimas Imobilizadas/química , Fluorescência , Glucanos/química , Limite de Detecção , Muramidase/química , Trealose/química
20.
Lab Chip ; 17(5): 943-950, 2017 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-28197602

RESUMO

We describe a versatile and simple method to perform sequential reactions on paper analytical devices by stacking dry pullulan films on paper, where each film contains one or more reagents or acts as a delay layer. Exposing the films to an aqueous solution of the analyte leads to sequential dissolution of the films in a temporally controlled manner followed by diffusive mixing of the reagents, so that sequential reactions can be performed. The films can be easily arranged for lateral flow assays or for spot tests (reactions take place sequentially in the z-direction). We have tested the general feasibility of the approach using three different model systems to demonstrate different capabilities: 1) pH ramping from low to high and high to low to demonstrate timing control; 2) rapid ready-to-use two-step Simon's assays on paper for detection of drugs of abuse utilizing a 2-layer stack containing two different reagents to demonstrate the ability to perform assays in the z-direction; and 3) sequential cell lysing and colorimetric detection of an intracellular bacterial enzyme, to demonstrate the ability of the method to perform sample preparation and analysis in the form of a spot assay. Overall, these studies demonstrate the potential of stacked pullulan films as useful components to enable multi-step assays on simple paper-based devices.


Assuntos
Glucanos/química , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodos , Papel , Dietilaminas/análise , Desenho de Equipamento , Escherichia coli/isolamento & purificação , Concentração de Íons de Hidrogênio , Indicadores e Reagentes/química
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