3D Paper-Based Origami Device for Programmable Multifold Analyte Preconcentration.

In analytical chemistry, preconcentration represents a critical step able to enhance the accuracy of detection; however, the experimental procedures needed to preconcentrate samples might be characterized by drawbacks regarding the whole analytical process, e.g., being complex, invasive, and/or time-consuming. In this study, a novel 3D paper-based origami device is introduced for multifold analyte preconcentration. Leveraging the benefits of paper-based substrates, the proposed architecture boosts sample preconcentration while minimizing time and tasks for measurements, solely by exploiting the porous and versatile nature of paper-based substrates. In comparison with other paper-based approaches reported in the literature for preconcentration, the present architecture offers the ability to be programmed for obtaining the needed sensitivity increase without sacrificing measurement time. To demonstrate the efficacy of the novel approach, the 3D paper-based origami device was deeply characterized, including the most relevant parameters, i.e., disk size and number, unfolding time, and volume, and subsequently applied for the preconcentration and the detection of various analytes in real matrices, namely, mercury in tap water and glucose in sweat, resulting in a 400% and 300% sensitivity enhancement, respectively. This innovative preconcentration tool addresses the limitations of existing conventional methods, providing increased sensitivity without the use of expensive and time-consuming procedures through only exploiting the intrinsic properties of paper-based substrates and a rationale design. The proposed architecture emerges as a universal tool to be adopted and programmed for various analytical systems and fields of application.

Remediate and sense: alginate beads empowered by portable electrochemical strips for copper ion removal and detection at environmental sites.

The contamination of environmental sites due to the presence of persistent species represents an important issue to be tackled. In particular, the presence of high levels of metals in soil and surface water is more frequent. One of the metals that sometimes exceeds the permissible limit set by regulatory authorities is copper. For instance, copper-based fungicides are widely used in viticulture. However, copper ions remain in soil and can enter the food chain, posing threats to human health and environmental safety. Although the rapid detection of copper ions using portable sensors is effective in enhancing early warning, it sometimes solves only half of the problem as remediation is not considered. In this paper, we present a novel integrated/portable approach that merges the remediation and sensing of metals by proposing a remediate-and-sense concept. In order to realize this concept, alginate beads were coupled with printed electrochemical strips for on-site copper detection. Within the same architecture, alginate beads were used to remove copper ions from the soil, and printed electrochemical strips were used to evaluate the efficacy of remediation at the point of need. The concept was applied towards soil containing copper ions at the parts per billion level; with few alginate beads and in the absence of additional species, copper ions were quantitatively removed from the matrix; and 3D printing allowed us to combine the printed strips and spheres within a unique tool. The architecture was optimized and the results were compared to inductively coupled plasma-mass spectrometry (ICP-MS) measurements with a recovery percentage of 90%-110%. It should be noted that this novel portable approach may be applied to other pollutants, opening new possibilities for integrated remediation and sensing.

Design and Validation of a Three-Dimensional Printer-Based System Enabling Rapid, Low-Cost Construction of the Biosensing Areas of Lateral Flow Devices for Immunoassays and Nucleic Acid Assays.

The COVID-19 pandemic proved the great usefulness of lateral flow tests as self- and rapid tests. The rapid expansion of this field requires the design and validation of novel, affordable, and versatile technologies for the easy fabrication of a variety of lateral flow devices. In the present work, we have developed a new, simple, and cost-effective system for the dispensing of reagents on the membranes of lateral flow devices to be used for research purposes. The 3D printing technology is integrated, for the first time, with simple and inexpensive tools such as a technical pen and disposable pipet tips for the construction of the test and the control areas of the devices. We also used this system for the automated fabrication of spots on the membrane for multiplex analysis. The devices were applied for the detection of proteins/antibodies and single- and double-stranded DNA targets. Also, devices with multiple biosensing areas on the membrane were constructed for the simultaneous detection of different analytes. The proposed system is very simple, automated, and inexpensive and has provided rapid and reproducible construction of lateral flow devices. Compared to a commercially available automated dispenser, the devices showed similar detection capabilities and reproducibility in various real samples. Moreover, contrary to the existing dispensers, the proposed system does not require any gas or costly precision pumps and syringes for the deposition. In conclusion, the developed 3D printer-based system could be an extremely useful alternative for research laboratories for the construction of lateral flow devices of various assay configurations.

A universal lateral flow assay for microRNA visual detection in urine samples.

MicroRNAs (miRNAs) have been emerged as novel and significant biomarkers in liquid biopsy that can be found in different body fluids. Several techniques have been developed and applied for miRNAs analysis, including nucleic acid-based amplification methods, next generation sequencing, DNA microarrays and new genome-editing methods. These methods, however, are time-consuming and require expensive instruments and specially trained personnel. Biosensors, on the other hand, are alternative and valuable analytical/diagnostic tools due to their simplicity, cost-effectiveness, rapid analysis and ease of use. Several biosensors, especially nanotechnology-based ones, have been developed for miRNA analysis that are based either on target amplification or signal amplification and target re-cycling for sensitive detection. At this point of view, we have introduced a new and universal lateral flow assay in combination with reverse transcription - polymerase chain reaction (RT-PCR) and gold nanoparticles as reporters for the detection of miR-21 and miR-let-7a in human urine. It is the first time that such a biosensor has been applied to the detection of microRNAs in urine. As low as 102-103 copies of miR-21 and 102--104 copies of miR-let-7a added in urine were detectable by the proposed lateral flow assay with great specificity and repeatability (%CVs <4.5%).

Rapid Multiplex Strip Test for the Detection of Circulating Tumor DNA Mutations for Liquid Biopsy Applications.

In the era of personalized medicine, molecular profiling of patient tumors has become the standard practice, especially for patients with advanced disease. Activating point mutations of the KRAS proto-oncogene are clinically relevant for many types of cancer, including colorectal cancer (CRC). While several approaches have been developed for tumor genotyping, liquid biopsy has been gaining much attention in the clinical setting. Analysis of circulating tumor DNA for genetic alterations has been challenging, and many methodologies with both advantages and disadvantages have been developed. We here developed a gold nanoparticle-based rapid strip test that has been applied for the first time for the multiplex detection of KRAS mutations in circulating tumor DNA (ctDNA) of CRC patients. The method involved ctDNA isolation, PCR-amplification of the KRAS gene, multiplex primer extension (PEXT) reaction, and detection with a multiplex strip test. We have optimized the efficiency and specificity of the multiplex strip test in synthetic DNA targets, in colorectal cancer cell lines, in tissue samples, and in blood-derived ctDNA from patients with advanced colorectal cancer. The proposed strip test achieved rapid and easy multiplex detection (normal allele and three major single-point mutations) of the clinically relevant KRAS mutations in ctDNA in blood samples of CRC patients with high specificity and repeatability. This multiplex strip test represents a minimally invasive, rapid, low-cost, and promising diagnostic tool for the detection of clinically relevant mutations in cancer patients.

Smartphone-based chemiluminometric hybridization assays and quantitative competitive polymerase chain reaction.

The present report introduces the smartphone as a simple, low-cost detector/imager for chemiluminometric hybridization assays and quantitative competitive polymerase chain reaction (QCPCR). In QCPCR the amplification products from the target and the competitor DNA have identical sizes but differ in a short sequence flanked by the primers. The products are hybridized with their cognate oligonucleotide probes, captured on microtiter wells and detected via an enzyme-catalyzed chemiluminogenic reaction using the smartphone as a detector/imager. We provide, for the first time, data on: (a) the detectability, analytical range and reproducibility of smartphone-based chemiluminometric hybridization assays of double stranded amplification products, (b) the comparison of smartphone-based detection with a conventional digital camera and a luminometer, and (c) the detectability, analytical range and reproducibility of smartphone-based QCPCR in terms of the number of copies of input target sequences in the sample prior to amplification. The limits of detection of the DNA hybridization assay based on the smartphone, digital camera and luminometer were 1.6, 2.4 and 1 pmol L-1. Smartphone-based QCPCR showed an analytical range from 137 to 9 × 105 copies of target DNA.

Advances in microRNA analysis.

MicroRNAs (miRNAs) are single-stranded noncoding RNA molecules that act as key regulators of mRNA expression and are emerging biomarkers for disease. Their small size (18-25 nt) presents challenges to molecular recognition, labeling, and signal generation. Recent research activity in this field has aimed at the development of methods for miRNA quantification that combine high detectability, broad dynamic range, practicality, multiplexity, and low cost for prospective applications in diagnostic laboratories. This review article covers the most recent advances in microRNA analysis.