Three-Dimensional-Printed Instrument for Isothermal Nucleic Acid Amplification with Real-Time Colorimetric Imaging.

Isothermal amplification methods have become popular in research due to the simplicity of the technology needed to run the reactions. Specifically, loop-mediated isothermal amplification (LAMP) has been widely used for various applications since first reported in 2000. LAMP reactions are commonly monitored with the use of colorimetry. Although color changes associated with positive amplification are apparent to the naked eye, this detection method is subjective due to inherent differences in visual perception from person to person. The objectivity of the colorimetric detection method may be improved by programmed image capture over time with simultaneous heating. As such, the development of a novel, one-step, automated, and integrated analysis system capable of performing these tasks in parallel is detailed herein. The device is adaptable to multiple colorimetric dyes, cost-effective, 3D-printed for single-temperature convective heating, and features an easy-to-use LabVIEW software program developed for automated image analysis. The device was optimized and subsequently validated using four messenger-RNA targets and mock forensic samples. The performance of our device was determined to be comparable to that of a conventional thermal cycler and smartphone image analysis, respectively. Moreover, the outlined system is capable of objective colorimetric analysis, with exceptional throughput of up to 96 samples at once.

Rapid, inexpensive fabrication of electrophoretic microdevices for fluorescence detection.

The laser print, cut, and laminate (PCL) method for microfluidic device fabrication can be leveraged for rapid and inexpensive prototyping of electrophoretic microchips useful for optimizing separation conditions. The rapid prototyping capability allows the evaluation of fluidic architecture, applied fields, reagent concentrations, and sieving matrix, all within the context of using fluorescence-compatible substrates. Cyclic olefin copolymer and toner-coated polyethylene terephthalate (tPeT) were utilized with the PCL technique and bonding methods optimized to improve device durability during electrophoresis. A series of separation channel designs and centrifugation conditions that provided successful loading of sieving polymer in less than 3 min was described. Separation of a 400-base DNA sizing ladder provided calculated base resolution between 3 and 4 bases, a greater than 18-fold improvement over separations on similar substrates. Finally, the accuracy and precision capabilities of these devices were demonstrated by separating and sizing DNA fragments of 147 and 167 bases as 148.62 ± 2 and 166.48 ± 3 bases, respectively.

Optimization of novel loop-mediated isothermal amplification with colorimetric image analysis for forensic body fluid identification.

Accurate presumptive and confirmatory test use for forensic body fluid identification is essential for gaining contextual information for crime scene investigators. Loop-mediated isothermal amplification (LAMP) is an ideal method for forensic body fluid identification because it is highly specific and generates multi-sized amplicon DNA, and successful amplification results can be read out colorimetrically. Here, we show preliminary data on a LAMP method that rapidly identifies body fluids including venous blood, semen, and saliva, based on colorimetric response and image analysis. The method is designed for easy implementation into forensic casework protocols with minimal disruption to DNA analysis. LAMP naturally increases target specificity due to the use of multiple primers for one target and mRNA targets were used for tissue and human specificity. With colorimetric detection as an inherent part of LAMP, samples that are positive or negative for any of the body fluids are readily identified by image capture and analysis, thus eliminating subjectivity. Results show by using the 3D-printed imaging system specific color ranges can be set for easy determination of body fluids. The resulting color change can be seen in <30 min using a universal temperature and primer concentration for all body fluids. This simple method and imaging system allow for minimal hands-on time with objective image analysis and presents a pathway for creating a new potential method for forensic body fluid identification.

Digital postprocessing and image segmentation for objective analysis of colorimetric reactions.

Recently, there has been an explosion of scientific literature describing the use of colorimetry for monitoring the progression or the endpoint result of colorimetric reactions. The availability of inexpensive imaging technology (e.g., scanners, Raspberry Pi, smartphones and other sub-$50 digital cameras) has lowered the barrier to accessing cost-efficient, objective detection methodologies. However, to exploit these imaging devices as low-cost colorimetric detectors, it is paramount that they interface with flexible software that is capable of image segmentation and probing a variety of color spaces (RGB, HSB, Y'UV, L*a*b*, etc.). Development of tailor-made software (e.g., smartphone applications) for advanced image analysis requires complex, custom-written processing algorithms, advanced computer programming knowledge and/or expertise in physics, mathematics, pattern recognition and computer vision and learning. Freeware programs, such as ImageJ, offer an alternative, affordable path to robust image analysis. Here we describe a protocol that uses the ImageJ program to process images of colorimetric experiments. In practice, this protocol consists of three distinct workflow options. This protocol is accessible to uninitiated users with little experience in image processing or color science and does not require fluorescence signals, expensive imaging equipment or custom-written algorithms. We anticipate that total analysis time per region of interest is ~6 min for new users and <3 min for experienced users, although initial color threshold determination might take longer.

Comparative Evaluation and Quantitative Analysis of Loop-Mediated Isothermal Amplification Indicators.

Loop-mediated isothermal amplification (LAMP) as a diagnostic tool is rapidly gaining recognition and maturity. Among various advantages over traditional polymerase chain reaction, the ability to visually detect amplification by the incorporation of colorimetric indicators is one of its most unique features. There is an overwhelming variety of LAMP indicators in the literature, yet a comprehensive comparative study is lacking. This study evaluates the use of hydroxynaphthol blue, phenol red, calcein, leuco crystal violet, malachite green, and a fluorescent dye for visual detection. A method for objective quantitative analysis using ImageJ is described that is readily implemented in standard and microfluidic workflows. The work here also includes the largest inter-reader variability study involving 24 participants to evaluate these indicators. We found inaccuracies in visual assessment as bias and/or individual-based perception can exist, solidifying the need for objective analysis. There was not a "universal" indicator, although considerations in sample preparation, storage, and applicability are discussed in length.

Automated microchannel alignment using innate opto-signature for microchip electrophoresis.

In laser-induced fluorescence (LIF) detection, optimal alignment is essential in maximizing the fluorescent signal and, hence, detection sensitivity. Micro-total analysis systems (µTAS) involving microchip electrophoresis (ME) are challenged with alignment of the optics to the separation channel each run due to the single-use nature. Furthermore, µTAS devices that are designed to operate autonomously and by non-experts face additional challenges in performing alignment with micrometer resolution without human intervention. As part of the development of a total DNA analysis system, we set out to develop an automated alignment (AA) method to locate a 50-by-50 µm separation channel on a freely rotating microfluidic device in the absence of a fluorescent dye, accomplished without additional hardware. We detail the innate fluorescent signature attainable from laser excitation and the optimization of the algorithm to achieve AA at 84.6% success rate from 26 microchips. This AA method was a key element in realizing complete automation of the DNA analysis process in order to advance our instrument to a technology readiness level of 7. This is the first description of an AA method for ME (and centrifugal ME) with the purpose of providing transparent technical details to bridge the gap from 'fully integrated' to 'fully automated' instruments for point-of-detection, sample in-answer-out use cases. Written in the context of a forensic application, the AA method is adaptable for a wide range of bioanalytical applications involving LIF detection.

Rapid sperm lysis and novel screening approach for human male DNA via colorimetric loop-mediated isothermal amplification.

Correct identification of probative samples is the first crucial step in the analysis of sexual assault kits (SAKs). We report a nucleic acid-based approach, as an alternative to the widely utilized p30 assay, to screening male DNA from SAKs collected from female victims by combining a rapid lysis protocol with an isothermal amplification method. The enzymatic lysis protocol efficiently digests biological material to release nuclear DNA in 10 min in a single closed tube, including resilient cell types such as sperm cells. The amplification and detection of human male specific DNA is achieved through loop-mediated isothermal amplification (LAMP) accompanied with hydroxynaphthol blue, a colorimetric indicator, producing a visually-distinctive color change in the presence of male DNA. The Y-screen approach demonstrated high specificity to human male DNA, can reliably detect target DNA as low as 50 pg, and correctly identified all probative samples from 14 single-blind mock sexual assault samples. In contrast with the widely used p30 assay which requires at least 2 h incubation time and manual application to a lateral flow pad, this Y-screen assay can be completed in half the time, and can be performed in a 96-well format without the need for a fluorescence detector, making facile high-throughput sample screening possible.