Point-of-care testing (POCT) with the advantages of speed, simplicity, and low cost, as well as no need for instrumentation, is critical for the measurement of analytes in a variety of environments lacking access to laboratory infrastructure. In the present study, a hydrogel pressure-based assay for quantitative POCT was developed by integrating a target-responsive hydrogel with pressuremeter readout. The target-responsive hydrogels were constructed with DNA grafted linear polyacrylamide and the cross-linking DNA for selective target recognition. The hydrogel response to the target substance allows release of the preloaded Pt nanoparticles, which have good stability and excellent catalytic ability for decomposing H2O2 to O2. Then, the generated O2 in a sealed environment leads to significant pressure increase, which can be easily read out by a handheld pressuremeter. Using this target-responsive hydrogel pressure-based assay, portable and highly sensitive detection of cocaine, ochratoxin A, and lead ion were achieved with excellent accuracy and selectivity. With the advantages of portability, high sensitivity, and simple sample processing, the hydrogel pressure-based assay shows great potential for quantitative POCT of a broad range of targets in resource-limited settings.Keywords: gas generation; pressuremeter readout; Pt nanoparticles; quantitative point-of-care testing; target-responsive hydrogel;

Aflatoxin B1 (AFB1), as the secondary metabolite of molds, is the most predominant and toxic mycotoxin that seriously threatens the health of humans and animals. In this work, an AFB1-responsive hydrogel was synthesized for highly sensitive and portable detection of AFB1. The AFB1-responsive hydrogel was prepared using an AFB1 aptamer and its two short complementary DNA strands as cross-linkers. For visual detection of AFB1, the hydrogel is preloaded with gold nanoparticles (AuNPs). Upon introduction of AFB1, the AFB1 aptamer binds with AFB1, leading to the disruption of the hydrogel and release of the AuNPs with a distinct color change of the supernatant from colorless to red. In order to lower the detection limit and extend the method to quantitative analysis, a distance-readout volumetric bar chart chip (V-chip) was combined with an AFB1-responsive hydrogel preloaded with platinum nanoparticles (PtNPs). In the presence of AFB1, the hydrogel collapses and releases PtNPs which can catalyze the decomposition of H2O2 to generate O2. The increasing gas pressure moves a red ink bar in the V-chip and provides a quantitative relationship between the distance and the concentration of AFB1. The method was applied for detection of AFB1 in beer, with a detection limit of 1.77 nM (0.55 ppb) where an immunoaffinity column (IAC) of AFB1 was used to cleanup and pre-concentrate the sample, which satisfies the testing requirement of 2.0 ppb set by the European Union. The combination of an AFB1-responsive hydrogel with a distance-based readout V-chip offers a user-friendly POCT device, which has great potential for rapid, portable, selective, and quantitative detection of AFB1 in real samples to ensure food safety and avoid subsequent economic losses.

Equipment-free devices with quantitative readout are of great significance to point-of-care testing (POCT), which provides real-time readout to users and is especially important in low-resource settings. Among various equipment-free approaches, distance-based visual quantitative detection methods rely on reading the visual signal length for corresponding target concentrations, thus eliminating the need for sophisticated instruments. The distance-based methods are low-cost, user-friendly and can be integrated into portable analytical devices. Moreover, such methods enable quantitative detection of various targets by the naked eye. In this review, we first introduce the concept and history of distance-based visual quantitative detection methods. Then, we summarize the main methods for translation of molecular signals to distance-based readout and discuss different microfluidic platforms (glass, PDMS, paper and thread) in terms of applications in biomedical diagnostics, food safety monitoring, and environmental analysis. Finally, the potential and future perspectives are discussed.

Abnormal DNA methylation patterns caused by altered DNA methyltransferase (MTase) activity are closely associated with cancer. Herein, using DNA adenine methylation methyltransferase (Dam MTase) as a model analyte, we designed an allosteric molecular beacon (aMB) for sensitive detection of Dam MTase activity. When the specific site in an aMB is methylated by Dam MTase, the probe can be cut by the restriction nuclease DpnI to release a fluorophore labeled aptamer specific for streptavidin (SA) which will bind to SA beads to generate highly fluorescent beads for easy signal readout by a microscope or flow cytometer. However, aMBs maintain a hairpin structure without the binding ability to SA beads in the absence of Dam MTase, leading to weakly fluorescent SA beads. Unlike the existing signal amplified assays, our method is simpler and more convenient. The high performance of the aptamer and the easy bead separation process make this probe superior to other methods for the detection of MTase in complex biological systems. Overall, the proposed method with a detection limit of 0.57 U mL−1 for Dam MTase shows great potential for further applications in the detection of other MTases, screening of MTase inhibitors, and early diagnosis of cancer.

•We constructed a hydrogel–μPAD system for POCT.•A glucoamylase-trapped aptamer-crosslinked hydrogel was used for molecular recognition.•Cascade enzymatic reactions were integrated for signal amplification•The system allows semiquantitation by naked eyes and quantitation by handheld devicesµPADs were used for portable readout.Paper based microfluidics (µPADs) with advantages of portability, low cost, and ease of use have attracted extensive attention. Here we describe a novel method that integrates glucoamylase-trapped aptamer-crosslinked hydrogel for molecular recognition with cascaded enzymatic reactions for signal amplification and a µPAD for portable readout. Upon target introduction, the hydrogel decomposes to release glucoamylase, which catalyzes the hydrolysis of amylose to produce a large amount of glucose. With a simple folding of the µPAD, the sample solution containing glucose product wicks and diffuses in parallel to each test-zone to carry out homogeneous assays, where glucose is used to produce I2 for brown color visualization through multiple enzymatic and chemical cascade reactions. Through color gradient changes based on different concentrations of the target, a semiquantitative assay is achieved by the naked eye, and quantitation can be obtained by handheld devices. Detection of cocaine in buffer and urine was performed to demonstrate the utility of the hydrogel–µPAD system. More importantly, the hydrogel–µPAD system can be extended to the detection of various targets by incorporating the corresponding aptamer into the hydrogel. The hydrogel–µPAD system reported here provides a new platform for portable, disposable and visual detection of a wide range of targets.

•We constructed a DNAzyme hydrogel system for portable detection of Uranium.•UO22+ dependent DNAzyme crosslinked hydrogel was designed and synthesized.•By encapsulating AuNPs inside hydrogel, UO22+ can be visually detected by naked eyes.•Portable quantitative detection was achieved by encapsulating PtNPs with V-Chip.Due to uranium's increasing exploitation in nuclear energy and its toxicity to human health, it is of great significance to detect uranium contamination. In particular, development of a rapid, sensitive and portable method is important for personal health care for those who frequently come into contact with uranium ore mining or who investigate leaks at nuclear power plants. The most stable form of uranium in water is uranyl ion (UO22+). In this work, a UO22+ responsive smart hydrogel was designed and synthesized for rapid, portable, sensitive detection of UO22+. A UO22+ dependent DNAzyme complex composed of substrate strand and enzyme strand was utilized to crosslink DNA-grafted polyacrylamide chains to form a DNA hydrogel. Colorimetric analysis was achieved by encapsulating gold nanoparticles (AuNPs) in the DNAzyme-crosslinked hydrogel to indicate the concentration of UO22+. Without UO22+, the enzyme strand is not active. The presence of UO22+ in the sample activates the enzyme strand and triggers the cleavage of the substrate strand from the enzyme strand, thereby decreasing the density of crosslinkers and destabilizing the hydrogel, which then releases the encapsulated AuNPs. As low as 100 nM UO22+ was visually detected by the naked eye. The target-responsive hydrogel was also demonstrated to be applicable in natural water spiked with UO22+. Furthermore, to avoid the visual errors caused by naked eye observation, a previously developed volumetric bar-chart chip (V-Chip) was used to quantitatively detect UO22+ concentrations in water by encapsulating Au-Pt nanoparticles in the hydrogel. The UO22+ concentrations were visually quantified from the travelling distance of ink-bar on the V-Chip. The method can be used for portable and quantitative detection of uranium in field applications without skilled operators and sophisticated instruments.

A target-responsive aptamer-cross-linked hydrogel was designed and synthesized for portable and visual quantitative detection of the toxin Ochratoxin A (OTA), which occurs in food and beverages. The hydrogel network forms by hybridization between one designed DNA strand containing the OTA aptamer and two complementary DNA strands grafting on linear polyacrylamide chains. Upon the introduction of OTA, the aptamer binds with OTA, leading to the dissociation of the hydrogel, followed by release of the preloaded gold nanoparticles (AuNPs), which can be observed by the naked eye. To enable sensitive visual and quantitative detection, we encapsulated Au@Pt core–shell nanoparticles (Au@PtNPs) in the hydrogel to generate quantitative readout in a volumetric bar-chart chip (V-Chip). In the V-Chip, Au@PtNPs catalyzes the oxidation of H2O2 to generate O2, which induces movement of an ink bar to a concentration-dependent distance for visual quantitative readout. Furthermore, to improve the detection limit in complex real samples, we introduced an immunoaffinity column (IAC) of OTA to enrich OTA from beer. After the enrichment, as low as 1.27 nM (0.51 ppb) OTA can be detected by the V-Chip, which satisfies the test requirement (2.0 ppb) by the European Commission. The integration of a target-responsive hydrogel with portable enrichment by IAC, as well as signal amplification and quantitative readout by a simple microfluidic device, offers a new method for portable detection of food safety hazard toxin OTA.Keywords: DNA hydrogel; microfluidics; Ochratoxin A; visual detection

Based on the protective properties of carbon nanoparticles for aptamers against the digestion of nuclease, we have developed a nuclease-assisted target recycling signal amplification method for highly sensitive detection of biomolecules, such as ATP and kanamycin. The high binding specificity between aptamers and targets leads to excellent selectivity of the assay.

A photo-reactive functional labelling reagent, diazirine phosphoramidite, was designed and synthesized for easy and flexible site-specific labelling of oligonucleotides with the diazirine moiety. The new reagent allows facile photo-crosslinking of oligonucleotide with its interacting partner for a variety of applications, including tertiary structure determination, molecular interaction study and biomarker discovery.

A T7 exonuclease-assisted cyclic enzymatic amplification method (CEAM) was combined with rolling circle amplification (RCA) to develop a RCA–CEAM dual amplification method for ultrasensitive detection of microRNA with excellent selectivity.

Accurate sensing of the extracellular pH is a very important yet challenging task in biological and clinical applications. This paper describes the development of an amphiphilic lipid–DNA molecule as a simple yet useful cell-surface-anchored ratiometric fluorescent probe for extracellular pH sensing. The lipid–DNA probe, which consists of a hydrophobic diacyllipid tail and a hydrophilic DNA strand, is modified with two fluorescent dyes; one is pH-sensitive as pH indicator and the other is pH-insensitive as an internal reference. The lipid–DNA probe showed sensitive and reversible response to pH change in the range of 6.0–8.0, which is suitable for most extracellular studies. In addition, based on simple hydrophobic interactions with the cell membrane, the lipid–DNA probe can be easily anchored on the cell surface with negligible cytotoxicity, excellent stability, and unique ratiometric readout, thus ensuring its accurate sensing of extracellular pH. Finally, this lipid–DNA-based ratiometric pH indicator was successfully used for extracellular pH sensing of cells in 3D culture environment, demonstrating the potential applications of the sensor in biological and medical studies.Keywords: cell surface anchorage; extracellular pH sensing; lipid−DNA; ratiometric fluorescent probe

Because of the severe health risks associated with lead pollution, rapid, sensitive, and portable detection of low levels of Pb2+ in biological and environmental samples is of great importance. In this work, a Pb2+-responsive hydrogel was prepared using a DNAzyme and its substrate as cross-linker for rapid, sensitive, portable, and quantitative detection of Pb2+. Gold nanoparticles (AuNPs) were first encapsulated in the hydrogel as an indicator for colorimetric analysis. In the absence of lead, the DNAzyme is inactive, and the substrate cross-linker maintains the hydrogel in the gel form. In contrast, the presence of lead activates the DNAzyme to cleave the substrate, decreasing the cross-linking density of the hydrogel and resulting in dissolution of the hydrogel and release of AuNPs for visual detection. As low as 10 nM Pb2+ can be detected by the naked eye. Furthermore, to realize quantitative visual detection, a volumetric bar-chart chip (V-chip) was used for quantitative readout of the hydrogel system by replacing AuNPs with gold–platinum core–shell nanoparticles (Au@PtNPs). The Au@PtNPs released from the hydrogel upon target activation can efficiently catalyze the decomposition of H2O2 to generate a large volume of O2. The gas pressure moves an ink bar in the V-chip for portable visual quantitative detection of lead with a detection limit less than 5 nM. The device was able to detect lead in digested blood with excellent accuracy. The method developed can be used for portable lead quantitation in many applications. Furthermore, the method can be further extended to portable visual quantitative detection of a variety of targets by replacing the lead-responsive DNAzyme with other DNAzymes.

Based on backbone-modified molecular beacons and duplex-specific nuclease, we have developed a target recycling amplification method for highly sensitive and selective miRNA detection. The combination of a low fluorescence background of 2-OMe-RNA modified MB and nuclease-assisted signal amplification leads to ultrahigh assay sensitivity, and the powerful discriminating ability of MB enables the differentiation of highly similar miRNAs with one-base difference, both of which are of great significance to miRNA detection.

Epithelial cell adhesion molecule (EpCAM) is overexpressed in most solid cancers and is an ideal antigen for clinical applications in cancer diagnosis, prognosis, imaging, and therapy. Currently, most of the EpCAM-based diagnostic, prognostic, and therapeutic strategies rely on the anti-EpCAM antibody. However, the use of EpCAM antibody is restricted due to its large size and instability. In this study, we have successfully identified DNA aptamers that selectively bind human recombinant EpCAM protein. The aptamers can specifically recognize a number of live human cancer cells derived from breast, colorectal, and gastric cancers that express EpCAM but not bind to EpCAM-negative cells. Among the aptamer sequences identified, a hairpin-structured sequence SYL3 was optimized in length, resulting in aptamer sequence SYL3C. The Kd values of the SYL3C aptamer against breast cancer cell line MDA-MB-231 and gastric cancer cell line Kato III were found to be 38 ± 9 and 67 ± 8 nM, respectively, which are better than that of the full-length SYL3 aptamer. Flow cytometry analysis results indicated that the SYL3C aptamer was able to recognize target cancer cells from mixed cells in cell media. When used to capture cancer cells, up to 63% cancer cell capture efficiency was achieved with about 80% purity. With the advantages of small size, easy synthesis, good stability, high binding affinity, and selectivity, the DNA aptamers reported here against cancer biomarker EpCAM will facilitate the development of novel targeted cancer therapy, cancer cell imaging, and circulating tumor cell detection.

We have combined an allosteric molecular beacon for target recognition and guanine-rich DNAzyme for signal amplification to develop a new platform for visual detection of nucleic acids with single-base mismatch detection capability. The fully DNA-structured platform can undergo color change in response to target DNA/RNA, which enables sensitive and selective visual detection in biological samples.

Nucleic acid molecular probes (NAMPs) have been widely used in the sensing of various chemical and biological substances, as well as physical parameters. However, for traditional nucleic acid molecular probes, the stoichiometric 1:1 binding ratio limits the signal enhancement and thus the sensitivity of the assay. In order to overcome this problem, the cyclic enzymatic amplification method (CEAM) based on exonuclease III has been applied in optical and electrical detection of DNA, proteins and small molecules with excellent sensitivity, selectivity, versatility and simplicity. In this review, the working principle of CEAM is first introduced, followed by the applications of CEAM using different output signals for various analytes. Finally, experimental designs and procedures of CEAM are discussed in detail using displacing probes-based CEAM and linear molecular beacons (LMBs)-based CEAM as two examples.

A photo-reactive functional labelling reagent, diazirine phosphoramidite, was designed and synthesized for easy and flexible site-specific labelling of oligonucleotides with the diazirine moiety. The new reagent allows facile photo-crosslinking of oligonucleotide with its interacting partner for a variety of applications, including tertiary structure determination, molecular interaction study and biomarker discovery.

Based on the protective properties of carbon nanoparticles for aptamers against the digestion of nuclease, we have developed a nuclease-assisted target recycling signal amplification method for highly sensitive detection of biomolecules, such as ATP and kanamycin. The high binding specificity between aptamers and targets leads to excellent selectivity of the assay.

We have combined an allosteric molecular beacon for target recognition and guanine-rich DNAzyme for signal amplification to develop a new platform for visual detection of nucleic acids with single-base mismatch detection capability. The fully DNA-structured platform can undergo color change in response to target DNA/RNA, which enables sensitive and selective visual detection in biological samples.

Based on backbone-modified molecular beacons and duplex-specific nuclease, we have developed a target recycling amplification method for highly sensitive and selective miRNA detection. The combination of a low fluorescence background of 2-OMe-RNA modified MB and nuclease-assisted signal amplification leads to ultrahigh assay sensitivity, and the powerful discriminating ability of MB enables the differentiation of highly similar miRNAs with one-base difference, both of which are of great significance to miRNA detection.

A T7 exonuclease-assisted cyclic enzymatic amplification method (CEAM) was combined with rolling circle amplification (RCA) to develop a RCA–CEAM dual amplification method for ultrasensitive detection of microRNA with excellent selectivity.