Visualising Microbial Activity: Colorimetric Signalling Using E. coli with pH-Indicators and Chromogenic Substrates

Photosensitization, originated from the activation of triplet states, is the basis of many photodynamic applications, but often competes with a series of nonradiative processes. Herein, we communic...

In this work, patterned β‐GUS sensing chitosan hydrogels functionalized with three different colorimetric substrates were fabricated for the multiplexed detection of the enzyme β‐glucuronidase (β‐GUS), which is secreted by >98% of all known Escherichia coli (E. coli) strains. The immobilization of fluorogenic and chromogenic substrates in specified areas allows a spatially resolved readout of the corresponding colorimetric signal. The apparent initial rate of the β‐GUS induced cleavage of the reporter moieties of chitosan films functionalized with the chromogenic substrate 4‐nitrophenyl‐β‐D‐glucuronide (PNPG), the chromogenic substrate 5‐bromo‐4‐chloro‐3‐indolyl‐β‐D‐glucuronide (X‐Gluc) and the fluorogenic substrate 4‐methylumbelliferyl‐β‐D‐glucuronide (MUG) were analyzed spectroscopically and confirmed a detectable reaction within less than 60 min. Likewise the released dyes were observed in the patterns owing to different colors by naked eye detection under appropriate illumination in less than 80 min. Hence the presence of a characteristic enzyme secreted by E. coli bacteria was successfully detected by three independent sensing moieties, which is important to reduce false positives by introducing redundancy. Patterned enzyme sensing chitosan hydrogels, which are functionalized with different substrates, open the possibility for multiplexed bacteria detection and in the long run also the identification of different bacteria strains.

Paper-based colorimetric biosensors combine the use of paper with colorimetric signal detection. However, they usually demonstrate lower sensitivities because a signal amplification procedure has not been used. Stopping the reaction of colorimetric signal generation is often used in lab-based assays in order to amplify and stabilize the colorimetric signal for detection. In this study, the generation of a stopped colorimetric signal was examined for accurate and enhanced signal detection in paper-based biosensors. The colorimetric reaction in biosensors is usually based on the interaction between the enzyme horseradish peroxidase (HRP) and a selected chromogenic substrate. The two most commonly used HRP substrates, 3,3’,5,5’-tetramethylbenzidine (TMB) and 2’-azinobis (3-ethylbenzothiazoline-6-sulfonic-acid) (ABTS), were compared in terms of their ability to generate a stopped colorimetric signal on membrane. The stopped colorimetric signal was visible for TMB but not for ABTS. Moreover, the generation of stopped colorimetric signal was dependent on the presence of polyvinylidene-difluoride (PVDF) membrane as the separation layer. With PVDF the colorimetric signal (color intensity) was higher (TMB: 126 ± 6 and ABTS: 121 ± 9) in comparison to without PVDF (TMB: 110 ± 2 and ABTS: 102 ± 4). The TMB stopped colorimetric signal demonstrated a more stable signal detection with lower standard deviation values. To conclude, a stopped colorimetric signal can be generated in paper-based biosensors for enhanced and accurate signal detection.

In this work, we report the use of logic platforms and iron-doped carbon dots (Fe-CDs) as a promising peroxidase-like analogue for the qualitative quantification of hydrogen peroxide (H2O2). The synthesised Fe-CDs exhibited excellent peroxidase-like (POD-like) activity compared to carbon dots (CDs), which catalyzed the formation of blue oxidation product (ox-TMB) with maximum absorption at 652 nm from the chromogenic substrate 3,3′, 5,5′ - tetramethylbenzidine (TMB). Steady state kinetic analysis shows that Fe-CDs have strong POD-like activity. Using the POD-like activity of Fe-CDs, H2O2 can be specifically detected colorimetrically. Based on this, we constructed a logic platform for H2O2 colourimetric sensing using Fe-CDs and H2O2 as inputs and the colorimetric signal as output. The H2O2 concentration and absorbance values showed good linearity in the range of 60–800 μM (R2 = 0.9887) with a limit of detection (LOD) of 7.25 μM, and the current method was successfully applied to the detection in mouthwash samples and laboratory tap water with recoveries in the range of 96.39 %–104.01 %. Fe-CDs catalyse the production of reactive oxygen species from H2O2, which can kill both Gram-positive and Gram-negative bacteria, and Fe-CDs have a highly efficient antimicrobial effect compared to CDs. Fe-CDs can be as high as 99.89 % antibacterial against E. coli and 97 % against S. aureus. In conclusion, we used herbal dregs to prepare Fe-CDs antimicrobial agent, and by recycling herbal dregs, the resources were maximised, and the nanozymes can not only construct the logical platform for H2O2 colorimetric sensing, but also show good application prospects in antimicrobials.

An enzymatic activity regulation-based clusterzyme sensor array for high-throughput identification of heavy metal ions

Fe7S8 nanosheets- catalyzed colorimetric and fluorescence dual ratio sensing for high selectivity detection of ascorbic acid

Portable hydrogel kit driven by bimetallic carbon dots nanozyme for H2O2-self-supplying dual-modal monitoring of atmospheric CH3SH

A dual-mode fluorescent and colorimetric immunoassay based on in situ ascorbic acid-induced signal generation from metal-organic frameworks

Miniaturization of nucleic acid tests (NATs) into portable, inexpensive detection platforms may aid disease diagnosis in point-of-care (POC) settings. Colorimetric signals are ideal readouts for portable NATs, and it remains of high demand to develop color readouts that are simple, quantitative, and versatile. Thus motivated, we report a fast light-activated substrate chromogenic polymerase chain reaction (FLASH PCR) that uses DNA intercalating dyes (DIDs) to enable colorimetric nucleic acid detection and quantification. The FLASH system is established on our finding that DID-DNA intercalation can promote the rapid photooxidation of chromogenic substrates through light-induced production of singlet oxygen. Using this principle, we have successfully converted DID-based fluorescent PCR assays into colorimetric FLASH PCR. To demonstrate the practical applicability of FLASH PCR to POC diagnosis, we also fabricated two readout platforms, including a portable electronic FLASH reader and a paper-based FLASH strip. Using the FLASH reader, we were able to detect as low as 60 copies of DNA standards, a limit of detection (LOD) comparable with commercial quantitative PCR. The FLASH strip further enables the reader-free detection of PCR amplicons by converting the colorimetric signal into the visual measurement of distance as a readout. Finally, the practical applicability of the FLASH PCR was demonstrated by the detection and/or quantification of nucleic acid markers in diverse clinical and biological samples.

• A plasmonic photothermal colorimetric biosensor is developed for molecular diagnostic assays. • Simple point-of-care system without the need of bulky and expensive instrumentation. • Fast (< 20 min) and highly sensitive (63.7 aM) colorimetric method for nucleic acid detection. • Colorimetric signals can be easily obtained with the naked eye and quantified by using a spectrophotometer and smart phone. The development of efficient and sensitive diagnostic method with no need for bulky and expensive instrumentation is of great importance in early detection and large-scale prevention of various infectious disease. Herein, we introduce a plasmonic photothermal colorimetric PCR (PPT-cPCR) for fast, sensitive, cheap, and simple nucleic acid detection. Our platform utilizes ultrafast PCR amplification based on the photothermal effect of plasmonic magnetic nanoparticles and color change by oxidation of chromogenic substrates caused by the photocatalytic activity of fluorescent dyes. PPT-cPCR has successfully shown that DNA targets ranging from 0.5 ng μL −1 to 3 fg μL −1 can be visually detected within 20 min with the naked eye. Additionally, the quantitative information can be easily obtained by using a spectrophotometer and smart phone, and the limit of detection (2.00 fg μL -1 , 63.7 aM) based on UV–vis absorbance is comparable with conventional PCR, however the assay time is about 2∼3 times shorter for the PPT-cPCR and the assay method is cost-effective. The strategy that combines PPT-based PCR and colorimetric analysis will open new horizons for point-of-care biosensor development.

Bifunctional fluorescent silver-based peroxidase-mimetic nanozymes: A novel ratiometric fluorescence/colorimetric dual-signal system for dopamine detection.

Point-of-care nucleic acid diagnostics demand rapid, instrument-free detection with high sensitivity and specificity. While loop-mediated isothermal amplification (LAMP) enables rapid amplification, conventional colorimetric indicators generate false positives by responding to any DNA synthesis, not just target-specific products. We developed a dual-gated G-quadruplex DNAzyme-LAMP that integrates G-quadruplex DNAzyme formation into the loop primer architecture while blocking non-specific activation through locked nucleic acidstabilized probe design. This approach gates colorimetric signal generation to occur only when target amplicons displace a 3'-blocking strand, enabling sequence-specific positive signaling without sacrificing amplification kinetics. When tested with Hepatitis A virus, the assay detected as few as 12 copies per reaction, matching RT-qPCR sensitivity while providing unambiguous positive colorimetric readouts. Specificity was maintained even in the presence of a 109-fold excess non-target DNA. Importantly, the platform requires only inexpensive hemin and chromogenic substrates, avoiding the protein reagents, custom oligonucleotides, and cold-chain logistics that constrain existing sequence-specific platforms. By exploiting the universally adopted loop primer element, this platform offers a generalizable framework for reliable colorimetric detection suitable for resource-limited outbreak settings.

A nanozyme-based colorimetric sensor array as electronic tongue for thiols discrimination and disease identification

Excellent performance of Ag6Si2O7 oxidase mimic endowed by self-released ions for rapid colorimetric determination with reduced operational interference

Colorimetric sensor array based on Au2Pt nanozymes for antioxidant nutrition quality evaluation in food