DNA-Templated Nanofabrication of CdS-Au Nanoscale Schottky Contacts and Electrical Characterization.

DNA-templated nanofabrication presents an innovative approach to creating self-assembled nanoscale metal-semiconductor-based Schottky contacts, which can advance nanoelectronics. Herein, we report the successful fabrication of metal-semiconductor Schottky contacts using a DNA origami scaffold. The scaffold, consisting of DNA strands organized into a specific linear architecture, facilitates the competitive arrangement of Au and CdS nanorods, forming heterojunctions, and addresses previous limitations in low electrical conductance making DNA-templated electronics with semiconductor nanomaterials. Electroless gold plating extends the Au nanorods and makes the necessary electrical contacts. Tungsten electrical connection lines are further created by electron beam-induced deposition. Electrical characterization reveals nonlinear Schottky barrier behavior, with electrical conductance ranging from 0.5 × 10-4 to 1.7 × 10-4 S. The conductance of these DNA-templated junctions is several million times higher than with our prior Schottky contacts. Our research establishes an innovative self-assembly approach with applicable metal and semiconductor materials for making highly conductive nanoscale Schottky contacts, paving the way for the future development of DNA-based nanoscale electronics.

Leveraging the third dimension in microfluidic devices using 3D printing: no longer just scratching the surface.

3D printers utilize cutting-edge technologies to create three-dimensional objects and are attractive tools for engineering compact microfluidic platforms with complex architectures for chemical and biochemical analyses. 3D printing's popularity is associated with the freedom of creating intricate designs using inexpensive instrumentation, and these tools can produce miniaturized platforms in minutes, facilitating fabrication scaleup. This work discusses key challenges in producing three-dimensional microfluidic structures using currently available 3D printers, addressing considerations about printer capabilities and software limitations encountered in the design and processing of new architectures. This article further communicates the benefits of using three-dimensional structures, including the ability to scalably produce miniaturized analytical systems and the possibility of combining them with multiple processes, such as mixing, pumping, pre-concentration, and detection. Besides increasing analytical applicability, such three-dimensional architectures are important in the eventual design of commercial devices since they can decrease user interferences and reduce the volume of reagents or samples required, making assays more reliable and rapid. Moreover, this manuscript provides insights into research directions involving 3D-printed microfluidic devices. Finally, this work offers an outlook for future developments to provide and take advantage of 3D microfluidic functionality in 3D printing. Graphical abstract Creating three-dimensional microfluidic structures using 3D printing will enable key advances and novel applications in (bio)chemical analysis.

Past, current, and future roles of 3D printing in the development of capillary electrophoresis systems.

3D printing, an additive manufacturing technology, has made significant inroads into improving systems for bioanalysis in recent years. This approach is particularly powerful due to the ease and flexibility in rapidly creating novel and complex designs for analytical applications. As such, 3D printing offers an emerging technology for creating systems for electrophoretic analysis. Here, we review 3D printing work on improving and miniaturizing capillary electrophoresis (CE), emphasizing publications from 2019‒2022. We describe enabling uses of 3D printing in interfacing upstream sample preparation or downstream detection with CE. Recent developments in miniaturized CE enabled by 3D printing are also elaborated, including key areas where 3D printing could further improve over the current state-of-the-art. Lastly, we highlight promising future trends for using 3D printing in miniaturizing CE and the significant potential for innovative advancements. 3D printing is poised to play a key role in moving forward miniaturized CE in the coming years.

Improving droplet microfluidic systems for studying single bacteria growth.

Antimicrobial resistance remains a global threat with ~ 5 million deaths in 2019 alone and 10 million deaths projected every year by 2050. Current tools employed in the analysis of bacteria can be time inefficient, leading to delayed diagnosis and treatment. In this work, we develop a microfluidic setup capable of bacteria incubation and detection of growth in ~ 2 h. We fabricated polydimethylsiloxane (PDMS) microchips via soft lithography, enclosed microchannels by plasma bonding to glass, and utilized PDMS blocks for simplified connection of devices to a flow system. We generated uniform droplets enclosing zero, one or two bacteria within our devices, and incubated droplet-encapsulated bacteria with 100 × lower concentrations of a fluorescence probe of bacterial growth compared to prior work. We assessed bacterial growth via laser induced fluorescence after room temperature incubation for 2 h and obtained a range of signals corresponding to droplets with or without bacteria. Our devices allow for online droplet incubation, monitoring, detection, and tracking. Developing microfluidic chips for single bacteria studies will improve the analysis and treatment of antimicrobial resistance.

Monolithic affinity columns in 3D printed microfluidics for chikungunya RNA detection.

Mosquito-borne pathogens plague much of the world, yet rapid and simple diagnosis is not available for many affected patients. Using a custom stereolithography 3D printer, we created microfluidic devices with affinity monoliths that could retain, noncovalently attach a fluorescent tag, and detect oligonucleotide and viral RNA. We optimized the fluorescent binding and sample load times using an oligonucleotide sequence from chikungunya virus (CHIKV). We also tested the specificity of CHIKV capture relative to genetically similar Sindbis virus. Moreover, viral RNA from both viruses was flowed through capture columns to study the efficiency and specificity of the column for viral CHIKV. We detected ~107 loaded viral genome copies, which was similar to levels in clinical samples during acute infection. These results show considerable promise for development of this platform into a rapid mosquito-borne viral pathogen detection system.

Immunoaffinity monoliths for multiplexed extraction of preterm birth biomarkers from human blood serum in 3D printed microfluidic devices.

In an effort to develop biomarker-based diagnostics for preterm birth (PTB) risk, we created 3D printed microfluidic devices with multiplexed immunoaffinity monoliths to selectively extract multiple PTB biomarkers. The equilibrium dissociation constant for each monoclonal antibody toward its target PTB biomarker was determined. We confirmed the covalent attachment of three different individual antibodies to affinity monoliths using fluorescence imaging. Three different PTB biomarkers were successfully extracted from human blood serum using their respective single-antibody columns. Selective binding of each antibody toward its target biomarker was observed. Finally, we extracted and eluted three PTB biomarkers from depleted human blood serum in multiplexed immunoaffinity columns in 3D printed microfluidic devices. This is the first demonstration of multiplexed immunoaffinity extraction of PTB biomarkers in 3D printed microfluidic devices.

Advances in multiplex electrical and optical detection of biomarkers using microfluidic devices.

Microfluidic devices can provide a versatile, cost-effective platform for disease diagnostics and risk assessment by quantifying biomarkers. In particular, simultaneous testing of several biomarkers can be powerful. Here, we critically review work from the previous 4 years up to February 2021 on developing microfluidic devices for multiplexed detection of biomarkers from samples. We focus on two principal approaches: electrical and optical detection methods that can distinguish and quantify biomarkers. Both electrical and spectroscopic multiplexed detection strategies are being employed to reach limits of detection below clinical sample levels. Some of the most promising strategies for point-of-care assays involve inexpensive materials such as paper-based microfluidic devices, or portable and accessible detectors such as smartphones. This review does not comprehensively cover all multiplexed microfluidic biomarker studies, but rather provides a critical evaluation of key work and suggests promising prospects for future advancement in this field. Electrical and optical multiplexing are powerful approaches for microfluidic biomarker analysis.

3D-printed microchip electrophoresis device containing spiral electrodes for integrated capacitively coupled contactless conductivity detection.

In this work, we demonstrate for the first time the design and fabrication of microchip electrophoresis devices containing cross-shaped channels and spiral electrodes around the separation channel for microchip electrophoresis and capacitively coupled contactless conductivity detection. The whole device was prepared in a digital light processing-based 3D printer in poly(ethylene glycol) diacrylate resin. Outstanding X-Y resolution of the customized 3D printer ensured the fabrication of 40-µm cross section channels. The spiral channels were filled with melted gallium to form conductive electrodes around the separation channel. We demonstrate the applicability of the device on the separation of sodium, potassium, and lithium cations by microchip electrophoresis. Graphical abstract.

3D printed microfluidic device for automated, pressure-driven, valve-injected microchip electrophoresis of preterm birth biomarkers.

A 3D printed, automated, pressure-driven injection microfluidic system for microchip electrophoresis (µCE) of preterm birth (PTB)-related peptides and proteins has been developed. Functional microvalves were formed, either with a membrane thickness of 5 µm and a layer exposure time of 450 ms or with a membrane thickness of 10 µm and layer exposure times of 300-350 ms. These valves allowed for control of fluid flow in device microchannels during sample injection for µCE separation. Device design and µCE conditions using fluorescently labeled amino acids were optimized. A sample injection time of 0.5 s and a separation voltage of 450 V (460 V/cm) yielded the best separation efficiency and resolution. We demonstrated the first µCE separation with pressure-driven injection in a 3D printed microfluidic device using fluorescently labeled PTB biomarkers and 532 nm laser excitation. Detection limits for two PTB biomarkers, peptide 1 and peptide 2, for an injection time of 1.5 s were 400 pM and 15 nM, respectively, and the linear detection range for peptide 2 was 50-400 nM. This 3D printed microfluidic system holds promise for future integration of on-chip sample preparation processes with µCE, offering promising possibilities for PTB risk assessment.

Rapid and simple pressure-sensitive adhesive microdevice fabrication for sequence-specific capture and fluorescence detection of sepsis-related bacterial plasmid gene sequences.

Microbial resistance to currently available antibiotics poses a great threat in the global fight against infections. An important step in determining bacterial antibiotic resistance can be selective DNA sequence capture and fluorescence labeling. In this paper, we demonstrate the fabrication of simple, robust, inexpensive microfluidic devices for DNA capture and fluorescence detection of a model antibiotic resistance gene sequence. We laser micromachined polymethyl methacrylate microchannels and enclosed them using pressure-sensitive adhesive tapes. We then formed porous polymer monoliths with DNA capture probes in these microchannels and used them for sequence-specific capture, fluorescent labeling, and laser-induced fluorescence detection of picomolar (pM) concentrations of synthetic and plasmid antibiotic resistance gene targets. The relative fluorescence for the elution peaks increased with loaded target DNA concentration. We observed higher fluorescence signal and percent recovery for synthetic target DNA compared to plasmid DNA at the same loaded target concentration. A non-target gene was used for control experiments and produced < 3% capture relative to the same concentration of target. The full analysis process including device fabrication was completed in less than 90 min with a limit of detection of 30 pM. The simplicity of device fabrication and good DNA capture selectivity demonstrated herein have potential for application with processes for bacterial plasmid DNA extraction and single-particle counting to facilitate determination of antibiotic susceptibility. Graphical abstract.

3D Printed Microfluidic Devices for Solid-Phase Extraction and On-Chip Fluorescent Labeling of Preterm Birth Risk Biomarkers.

Solid-phase extraction (SPE) is a general preconcentration method for sample preparation that can be performed on a variety of specimens. The miniaturization of SPE within a 3D printed microfluidic device further allows for fast and simple extraction of analytes while also enabling integration of SPE with other sample preparation and separation methods. Here, we present the development and application of a reversed-phase lauryl methacrylate-based monolith, formed in 3D printed microfluidic devices, which can selectively retain peptides and proteins. The effectiveness of these SPE monoliths and 3D printed microfluidic devices was tested using a panel of nine preterm birth biomarkers of varying hydrophobicities and ranging in mass from 2 to 470 kDa. The biomarkers were selectively retained, fluorescently labeled, and eluted separately from the excess fluorescent label in 3D printed microfluidic systems. These are the first results demonstrating microfluidic analysis processes on a complete panel of preterm birth biomarkers, an important step toward developing a miniaturized, fully integrated analysis system.

Impact of Polymer-Constrained Annealing on the Properties of DNA Origami-Templated Gold Nanowires.

DNA origami-templated fabrication enables bottom-up fabrication of nanoscale structures from a variety of functional materials, including metal nanowires. We studied the impact of low-temperature annealing on the morphology and conductance of DNA-templated nanowires. Nanowires were formed by selective seeding of gold nanorods on DNA origami and gold electroless plating of the seeded structures. At low annealing temperatures (160 °C for seeded-only and 180 °C for plated), the wires broke up and separated into multiple, isolated islands. Through the use of polymer-constrained annealing, the island formation in plated wires was suppressed up to annealing temperatures of 210 °C. Four-point electrical measurements showed that the wires remained conductive after a polymer-constrained annealing at 200 °C.

Seeding, Plating and Electrical Characterization of Gold Nanowires Formed on Self-Assembled DNA Nanotubes.

Self-assembly nanofabrication is increasingly appealing in complex nanostructures, as it requires fewer materials and has potential to reduce feature sizes. The use of DNA to control nanoscale and microscale features is promising but not fully developed. In this work, we study self-assembled DNA nanotubes to fabricate gold nanowires for use as interconnects in future nanoelectronic devices. We evaluate two approaches for seeding, gold and palladium, both using gold electroless plating to connect the seeds. These gold nanowires are characterized electrically utilizing electron beam induced deposition of tungsten and four-point probe techniques. Measured resistivity values for 15 successfully studied wires are between 9.3 × 10-6 and 1.2 × 10-3 Ωm. Our work yields new insights into reproducible formation and characterization of metal nanowires on DNA nanotubes, making them promising templates for future nanowires in complex electronic circuitry.

3D Printed Microfluidic Devices for Microchip Electrophoresis of Preterm Birth Biomarkers.

This work demonstrates for the first time the creation of microchip electrophoresis devices with ∼50 µm cross-sectional dimensions by stereolithographic 3D printing and their application in the analysis of medically significant biomarkers related to risk for preterm birth (PTB). We determined that device current was linear with applied potential up to 800 V (620 V/cm). We optimized device and separation conditions using fluorescently labeled amino acids as a model system and compared the performance in our 3D printed microfluidic devices to that in other device materials commonly used for microchip electrophoresis analysis. We demonstrated for the first time microchip electrophoresis in a 3D printed device of three PTB biomarkers, including peptides and a protein, with suitable separation characteristics. Limits of detection for microchip electrophoresis in 3D printed microfluidic devices were also determined for PTB biomarkers to be in the high picomolar to low nanomolar range.

Analysis of thrombin-antithrombin complex formation using microchip electrophoresis and mass spectrometry.

Preterm birth (PTB) related health problems take over one million lives each year, and currently, no clinical analysis is available to determine if a fetus is at risk for PTB. Here, we describe the preparation of a key PTB risk biomarker, thrombin-antithrombin (TAT), and characterize it using dot blots, MS, and microchip electrophoresis (µCE). The pH for fluorescently labeling TAT was also optimized using spectrofluorometry and spectrophotometry. The LOD of TAT was measured in µCE. Lastly, TAT was combined with six other PTB risk biomarkers and separated in µCE. The ability to make and characterize TAT is an important step toward the development of an integrated microfluidic diagnostic for PTB risk.

3D printed microfluidic devices with immunoaffinity monoliths for extraction of preterm birth biomarkers.

Preterm birth (PTB) is defined as birth before the 37th week of pregnancy and results in 15 million early deliveries worldwide every year. Presently, there is no clinical test to determine PTB risk; however, a panel of nine biomarkers found in maternal blood serum has predictive power for a subsequent PTB. A significant step in creating a clinical diagnostic for PTB is designing an automated method to extract and purify these biomarkers from blood serum. Here, microfluidic devices with 45 µm × 50 µm cross-section channels were 3D printed with a built-in polymerization window to allow a glycidyl methacrylate monolith to be site-specifically polymerized within the channel. This monolith was then used as a solid support to attach antibodies for PTB biomarker extraction. Using these functionalized monoliths, it was possible to selectively extract a PTB biomarker, ferritin, from buffer and a human blood serum matrix. This is the first demonstration of monolith formation in a 3D printed microfluidic device for immunoaffinity extraction. Notably, this work is a crucial first step toward developing a 3D printed microfluidic clinical diagnostic for PTB risk.

Microchip electrophoresis separation of a panel of preterm birth biomarkers.

Preterm birth (PTB) is responsible for over one million infant deaths annually worldwide. Often, the first and only indication of PTB risk is the onset of early labor. Thus, there is an urgent need for an early PTB risk diagnostic that is inexpensive, reliable, and robust. Here, we describe the development of a microchip electrophoresis (µCE) method for separating a mixture of six PTB protein and peptide biomarkers present in maternal blood serum. µCE devices were photografted with a poly(ethylene glycol) diacrylate surface coating to regulate EOF and reduce nonspecific analyte adsorption. Separation conditions including buffer pH, buffer concentration, and applied electric field were varied to improve biomarker peak resolution while minimizing deleterious effects like Joule heating. In this way, it was possible to separate six PTB biomarkers, the first µCE separation of this biomarker panel. LODs were also measured for each of the six PTB biomarkers. In the future, this µCE separation can be integrated with upstream maternal blood serum sample preparation steps to yield a complete PTB risk diagnosis microdevice.

Four-Point Probe Electrical Measurements on Templated Gold Nanowires Formed on Single DNA Origami Tiles.

Bottom-up nanofabrication is increasingly making use of self-assembled DNA to fabricate nanowires and potential integrated circuits, although yields of such electronic nanostructures are inadequate, as is the ability to reliably make electrical measurements on them. In this paper, we report improved yields and unprecedented conductivity measurements for Au nanowires created on DNA origami tile substrates. We created several different self-assembled Au nanowire arrangements on DNA origami tiles that are approximately 70 nm × 90 nm, through anisotropic growth of Au nanorods attached to specific sites. Modifications to the tile design increased yields of the final desired nanostructures as much as 6-fold. In addition, we measured the conductivity of Au nanowires created on these DNA tiles (∼130 nm long, 10 nm diameter, and 40 nm spacing between measurement points) with a four-point measurement technique that utilized electron beam induced metal deposition to form probe electrodes. These nanowires formed on single DNA origami tiles were electrically conductive, having resistivities as low as 4.24 × 10-5 Ω m. This work demonstrates the creation and measurement of inorganic nanowires on single DNA origami tiles as a promising path toward future bottom-up fabrication of nanoelectronics.