Juan G. Santiago

Abstract Fluorescence-based CRISPR diagnostic assays have become a popular platform for nucleic acid detection due to their programmability, configurability, specificity, and compatibility with standard laboratory equipment. However, reported enzymatic kinetic rates and limits of detection for CRISPR trans- cleavage assays vary by several orders of magnitude across the literature. This variation in performance parameters is coupled with and exacerbated by inconsistent calibration, incomplete correction of measurement biases, and nonstandardized or incomplete data-analysis procedures. We present an experimental protocol and quantitative analysis framework for fluorescence-based enzyme assays using routine laboratory instrumentation, including thermocyclers and fluorescence microplate readers. Building on previous studies of CRISPR enzyme kinetics and fluorescence calibration, we describe procedures for flat-field and background correction; comprehensive fluorescence calibration including correction for inner-filter-effect; quantification and implications of reporter degradation; extraction of Michaelis-Menten kinetic parameters; and determination of assay limits of detection. We provide step-by-step experimental guidelines and open-source Python implementations for each stage of the workflow. Using representative Cas12 trans- cleavage datasets, we demonstrate that explicit fluorescence calibration and correction procedures substantially reduce systematic bias in measured kinetic rates and improve consistency between experiments. Our framework aims to establish standardized practices for quantitative fluorescence-based CRISPR assays and provides researchers with practical tools for reproducible kinetic characterization and rational assay design.

CRISPR-based diagnostics are powerful tools for nucleic acid detection due to their high specificity and programmability. However, assay sensitivity is often limited by the slow kinetics of the trans -cleavage reaction, which typically proceeds at a rate of ∼0.1 to 1 turnover per second. Here, we present a reaction-transport model and experimental study that analyze and accelerate this limiting step using electric-field-driven isotachophoresis (ITP). Building on the work of Ramachandran and Santiago, we develop a model that captures the coupling among ITP focusing, mixing, and preconcentration with CRISPR enzymatic reaction kinetics. Our analysis identifies two key regimes in ITP-coupled CRISPR reactions and derives analytical approximations for the limiting behaviors in each. Compared to a standard, well-mixed assay, we predict a 10- to 100-fold reduction in reaction duration using ITP. We validate the model with experiments across a range of target concentrations. Our work offers a quantitative framework for understanding and optimizing CRISPR trans -cleavage dynamics and provides guidance to design assays that use electric-field-mediated transport.

The development of microfluidic technologies has enabled chemical and biological analysis systems with increased functionality, complexity, and parallelization. These functionalities often drive the creation and control of complex and dynamic fluidic architectures. Introduced here is a class of microfluidic network based on isotachophoresis (ITP), an electrokinetic process that can extract and purify samples, selectively transport, mix, and aliquot (split) samples in a system with no moving parts. Presented is a theoretical framework to describe these networks. The framework relies on the coupling between a one-dimensional description of ITP and two-dimensional, transient graphs to describe the dynamic evolution of ITP networks. We leverage this framework to create numerical simulations of branched ITP circuits. We build, control, and experimentally study a variety of ITP networks. These systems automatically split and merge ITP zones, enabling complex sample manipulation with minimal external control. The model captures the experimentally observed sample dynamics. We demonstrate an example system where an ITP network is used to control and quantify parallel CRISPR-Cas enzymatic reactions. The methods described here are generally applicable to highly complex topologies and may offer a basis for easily reconfigurable, electric field-driven microfluidic systems. Networks generally offer broad potential for automated chemical and biochemical analysis and lab-on-a-chip integration.

The sensitivity of CRISPR-Cas systems used for molecular diagnostics remains a major bottleneck in the adoption of this technology. The vast majority of CRISPR-based assays use dually labeled, single-stranded reporters and fluorescence signal readouts to infer enzymatic activity and the presence (or absence) of target nucleic acid. The limit of detection of such assays is set by the kinetics of the Cas enzymes and a slow yet measurable increase in the fluorescence signal. We demonstrate here that the background signal and limits of detection of most assays are very likely limited by the degradation of reporter molecules. This degradation is dynamic and is not associated with enzymatic activity. We present theory and experiments to design and calibrate CRISPR assays. We introduce a new kinetic framework to account for the degradation of reporter molecules and derive a fundamental limit of detection for CRISPR-based assays. Our data show that Michaelis-Menten kinetics alone are insufficient to describe reporter (substrate) cleavage rates. The framework and techniques presented here should help reduce the frequency and magnitude of errors currently routinely made in quantifying CRISPR kinetics and interpreting CRISPR diagnostic fluorescence signals.

The dispersion behaviour of solutes in flow is crucial to the design of chemical separation systems and microfluidics devices. These systems often rely on coupled electroosmotic and pressure-driven flows to transport and separate chemical species, making the transient dispersive behaviour of solutes highly relevant. However, previous studies of Taylor dispersion in coupled electroosmotic and pressure-driven flows focused on the long-term dispersive behaviour and the associated analyses cannot capture the transient behaviour of solute. Further, the radial distribution of solute has not been analysed. In the current study, we analyse the Taylor dispersion for coupled electroosmotic and pressure-driven flows across all time regimes, assuming a low zeta potential (electric potential at the shear plane), the Debye–Hückel approximation and a finite electric double layer thickness. We first derive analytical expressions for the effective dispersion coefficient in the long-time regime. We also derive an unsteady, two-dimensional (radial and axial) solute concentration field applicable in the latter regime. We next apply Aris’ method of moments to characterise the unsteady propagation of the mean axial position and the unsteady growth of the variance of the solute zone in all time regimes. We benchmark our predictions with Brownian dynamics simulations across a wide and relevant dynamical regime, including various time scales. Lastly, we derive expressions for the optimal relative magnitudes of electroosmotic versus pressure-driven flow and the optimum Péclet number to minimise dispersion across all time scales. These findings offer valuable insights for the design of chemical separation systems, including the optimisation of capillary electrophoresis devices and electrokinetic microchannels and nanochannels.

Trapping, linearization, and imaging of single-molecule DNA are of broad interest to both biophysicists who study polymer physics and engineers who build nucleic acid analysis methods such as optical mapping. In this study, single DNA molecules in a neutral linear polymer solution are driven with an axial electric field through microchannels, and their dynamics are studied using fluorescence microscopy. Above a certain threshold electric field, individual DNA molecules become pinned to the channel walls at a vertex on each molecule and are stretched in the direction opposite to electric field. Upon removal of the electric field, pinned DNA molecules undergo relaxation within a few seconds to a Brownian coil around the vertex. After tens of seconds, DNA is released and free to diffuse and electromigrate. The method enables high-quality imaging of single-molecule DNA with high throughput using simple-to-fabricate fluidic structures. The conditions required for trapping dynamics, relaxation dynamics, and the repeatability of vertex pinning are analyzed. It is hypothesized that the neutral linear (non-cross-linked) polymers adsorb to the wall and form scaffolds that trap DNA. Potential hypotheses are discussed based on the empirical findings to explain potential physical mechanism of such unique trapping behavior in a non-crosslinked linear polymer solution.

Flowable carbon slurries are actively studied and under development for charge transport in various electrochemical systems including flow capacitors, capacitive deionization cells, semi-solid flow batteries, and lithium extraction. However, much less is known about in operando slurry flow dynamics and their corresponding effect on charge transport. We performed an experimental study of mixing and settling dynamics of slurry electrodes within an electrochemical flow cell during continuous operations. The electrochemical cell consisted of two horizontal co-flowing channels, separated by a cation-exchange membrane (CEM). We used high-speed optical imaging of planes parallel to gravity and simultaneous electrochemical measurements. At low flow rates, dense yet dynamic particle beds formed on the bottom electrode in each channel, which unexpectedly yielded the highest currents. This approach enables the operation of the flow cell at low system-average particle concentrations while leveraging gravity-driven particle settling to locally enhance carbon concentrations precisely at the current collector sites. Conversely, high flow rates were characterized by thin particle beds and well-mixed particle flows. In the latter case, the electrodes in closest proximity (located on either side of the CEM) achieved a current higher than the other electrode pairs. The observations have implications for slurry control and electrode designs in electrochemical systems.

This Feature Article reviews engineering guidelines for the design of CRISPR assays, including experimentally validated theoretical models and recommendations for experimental research practice and reporting. First, the state of the art of CRISPR kinetics studies is reviewed. Then presented is a summary of the existence and persistence of widespread gross errors in reports of kinetic rate constants of CRISPR-Cas enzymes, as well as the fact that many CRISPR studies provide insufficient data to check for consistency or assess calibration. Proper experimental procedures including signal calibration are critical to the assessment, design, and future development of CRISPR kinetics assays and CRISPR diagnostics. This review then presents guidelines for the calibration of fluorescence-based CRISPR assays. Fluorescence is the most common detection modality, and incorrect calibration is implicated in high-profile, gross errors in the field. Also presented is a review of enzymatic kinetic rates and reporter molecule degradation as the major factor limiting CRISPR assay sensitivity. Lastly, progress in, and criticism of, microfluidic applications of CRISPR assays is summarized.

within a total process time of 60 min in these experiments. Human serum samples processed without purification show no PCR amplification results. This integrated system demonstrates the powerful concept of integrating microfluidic structures into form factors compatible with the highly complex and sensitive operation of current off-the-shelf systems.

BACKGROUND Isotachophoresis (ITP) is a well-established electrokinetic method for separation and preconcentration of analytes. Several simulation tools for ITP have been published, but their use for experimental design is limited by the computational time for a single run and/or by the number of conditions that can be investigated per simulation run. A large fraction of the existing solvers also do not account for ionic strength effects, which can influence whether an analyte focuses in ITP. There is currently no publicly available tool for the easy and rapid design of ITP experiments. RESULTS We present a rapid, highly parallelized steady-state solver for the design of buffer electrolytes in ITP experiments. The tool is called Browser-based Electrolyte Analyses for ITP (BEAN). BEAN is designed to facilitate the evaluation and identification of functional buffer chemistries for ITP. Given a user-defined chemistry system, BEAN solves a set of coupled, non-linear integral conservation equations to determine whether a specific analyte is focused by the ITP system, and estimates quantities of interest in the design of related ITP processes. These quantities include zone concentrations, pH, and effective (observable) mobility values. BEAN also computes 972 variations of the specified ITP chemistry, including a broad range of buffer titrations and ion mobilities. All the calculations performed in BEAN include ionic strength and finite ionic radius effects, and the solver handles species with arbitrary valence. The tool further includes a searchable database of 521 commonly used electrolytes. BEAN is available at microfluidics.stanford.edu/bean. SIGNIFICANCE This study introduces a novel tool that integrates known ITP steady-state equations with a highly parallel computational framework, an electrolyte database, and a web-based interface. BEAN requires no license nor compilation, and its parallel computations are performed automatically without specific implementation needed from the user. This enables users to screen wide ranges of experimental conditions in the design of ITP experiments.

Trapping, linearization, and imaging of single molecule DNA is of broad interest to both biophysicists who study polymer physics and engineers who build nucleic acid analyzing methods such as optical mapping. In this study, single DNA molecules in a neutral linear polymer solution were driven with an axial electric field through microchannels and their dynamics were studied using fluorescence microscopy. We observed that above a threshold electric field, individual DNA molecules become pinned to the channel walls at a vertex on each molecule and are stretched in the direction opposite to the electric field. Upon removal of the electric field, pinned DNA molecules undergo relaxation within a few seconds to a Brownian coil around the vertex. After 10s of seconds, DNA is released and free to electromigrate. The method enables high quality imaging of single-molecule DNA with high throughput using simple-to-fabricate fluidic structures. We analyze the conditions needed for trapping, relaxation dynamics, and the repeatability of vertex pinning. We hypothesize DNA entangles with neutral linear polymers adsorbed to walls. We hypothesize that a sufficiently high electric force on the DNA is required to expel a hydration layer between the DNA and the wall-adsorbed neutral linear polymers. The elimination of the hydration layer may increase the friction between charged DNA and the uncharged polymer, promoting vertex pinning of DNA.

Infectious diseases such as tuberculosis, typhoid fever, or dengue remain a global threat.Rapid and accurate diagnostic tests facilitate early recognition and treatment of infectious diseases, enabling improved clinical care, and timely implementation of infection control and other public health measures.However, the vast majority of existing nucleic acid detection assays requires instrumentation that is not portable, and sample preparation and assay methods by trained technicians on specialized instruments in clinical laboratories.There is a critical need for the development of an inexpensive, fielddeployable device for accurate nucleic acid detection.[1]

Next-generation sequencing offers highly multiplexed and accurate detection of nucleic acid sequences but at the expense of complex workflows and high input requirements. The ease of use of CRISPR-Cas12 assays is attractive and may enable highly accurate detection of sequences implicated in, for example, cancer pathogenic variants. CRISPR assays often employ end-point measurements of Cas12 trans-cleavage activity after Cas12 activation by the target; however, end point-based methods can be limited in accuracy and robustness by arbitrary experimental choices. To overcome such limitations, we develop and demonstrate here an accurate assay targeting a mutation of the epidermal growth factor gene implicated in lung cancer (exon 19 deletion). The assay is based on characterizing the kinetics of Cas12 trans-cleavage to discriminate the mutant from wild-type targets. We performed extensive experiments (780 reactions) to calibrate key assay design parameters, including the guide RNA sequence, reporter sequence, reporter concentration, enzyme concentration, and DNA target type. Interestingly, we observed a competitive reaction between the target and reporter molecules that has important consequences for the design of CRISPR assays, which use preamplification to improve sensitivity. Finally, we demonstrate the assay on 18 tumor-extracted amplicons and 100 training iterations with 99% accuracy and discuss discrimination parameters and models to improve wild type versus mutant classification.

Various aspects of the present disclosure are directed toward methods and apparatuses for interacting a first liquid and a second liquid in one or more fluidic channels of a capillary structure. The methods and apparatuses can include providing at least one capillary barrier that positions a meniscus of the first liquid at a fluid-interface region using capillary forces within the capillary structure. Additionally, a path is provided along one of the channels for the second liquid to flow toward the fluid-interface region. Additionally, gas pressure is released, via a gas-outflow port, from the fluid-interface region while flow of the first liquid is arrested. Further, the first liquid and the second liquid contact in the fluid-interface region with the capillary barrier holding the first liquid at the fluid-interface region.

Understanding electrokinetic transport in nanochannels and nanopores is essential for emerging biological and electrochemical applications. The viscoelectric effect is an important mechanism implicated in the increase of local viscosity due to the polarization of a solvent under a strong electric field. However, most analyses of the viscoelectric effect have been limited to numerical analyses. In this work, we present a set of analytical solutions applicable to the physical description of viscoelectric effects in nanochannel electrokinetic systems. To achieve such closed-form solutions, we employ the Debye-Hückel approximation of small diffuse charge layer potentials compared to the thermal potential. We analyze critical parameters, including electroosmotic flow profiles, electroosmotic mobility, flow rate, and channel conductance. We compare and benchmark our analytical solutions with published predictions from numerical models. Importantly, we leverage these analytical solutions to identify essential thermophysical and nondimensional parameters that govern the behavior of these systems. We identify scaling parameters and relations among surface charge density, ionic strength, and nanochannel height.

Advective dispersion of solutes in long thin axisymmetric channels is important to the analysis and design of a wide range of devices, including chemical separation systems and microfluidic chips. Despite extensive analysis of Taylor dispersion in various scenarios, most studies focus on long-term dispersion behaviour and cannot capture the transient evolution of the solute zone across the spatial variations in the channel. In the current study, we analyse the Taylor–Aris dispersion for arbitrarily shaped axisymmetric channels. We derive an expression for solute dynamics in terms of two coupled ordinary differential equations, which allow prediction of the time evolution of the mean location and axial (standard deviation) width of the solute zone as a function of the channel geometry. We compare and benchmark our predictions with Brownian dynamics simulations for a variety of cases, including linearly expanding/converging channels and periodic channels. We also present an analytical description of the physical regimes of transient positive versus negative axial growth of solute width. Finally, to further demonstrate the utility of the analysis, we demonstrate a method to engineer channel geometries to achieve desired solute width distributions over space and time. We apply the latter analysis to generate a geometry that results in a constant axial width and a second geometry that results in a sinusoidal axial variance in space.

We present the development and demonstration of a neural network (NN) model for fast and accurate prediction of whether or not a chosen analyte is focused by an isotachophoresis (ITP) buffer system. The NN model is useful in the rapid evaluation of possible ITP chemistries applicable to analytes of interest. We trained and tested the NN model for univalent species based on extensive data sets of over 10,000 anionic and 10,000 cationic ITP simulations. The NN model uses as inputs the mobilities and the acid dissociation constants of leading electrolyte ion, trailing electrolyte ion, counterion, and a single analyte as well as the leading-to-counterion concentration ratio of the leading zone. The output then indicates whether the chosen electrolyte system yields stable ITP focusing of the analyte. The prediction accuracy of the NN model is over 97.7%. We demonstrate the applicability of the NN by validating its predictions with reported experimental data for anionic and cationic ITP. We have packaged the NN model in a free, web-based application named IONN (isotachophoresis on neural network), which can be used to rapidly screen ITP electrolyte systems.

Clinical trials have become the primary mechanism to validate process improvements in oncology clinical practice. Over the past two decades there have been considerable process improvements in the practice of radiation oncology within the structure of a modern department using advanced technology for patient care. Treatment planning is accomplished with volume definition including fusion of multiple series of diagnostic images into volumetric planning studies to optimize the definition of tumor and define the relationship of tumor to normal tissue. Daily treatment is validated by multiple tools of image guidance. Computer planning has been optimized and supported by the increasing use of artificial intelligence in treatment planning. Informatics technology has improved, and departments have become geographically transparent integrated through informatics bridges creating an economy of scale for the planning and execution of advanced technology radiation therapy. This serves to provide consistency in department habits and improve quality of patient care. Improvements in normal tissue sparing have further improved tolerance of treatment and allowed radiation oncologists to increase both daily and total dose to target. Radiation oncologists need to define a priori dose volume constraints to normal tissue as well as define how image guidance will be applied to each radiation treatment. These process improvements have enhanced the utility of radiation therapy in patient care and have made radiation therapy an attractive option for care in multiple primary disease settings. In this chapter we review how these changes have been applied to clinical practice and incorporated into clinical trials. We will discuss how the changes in clinical practice have improved the quality of clinical trials in radiation therapy. We will also identify what gaps remain and need to be addressed to offer further improvements in radiation oncology clinical trials and patient care.

Reviewed are nucleic acid detection assays that incorporate clustered regularly interspaced short palindromic repeats (CRISPR)-based diagnostics and microfluidic devices and techniques. The review serves as a reference for researchers who wish to use CRISPR-Cas systems for diagnostics in microfluidic devices. The review is organized in sections reflecting a basic five-step workflow common to most CRISPR-based assays. These steps are analyte extraction, pre-amplification, target recognition, transduction, and detection. The systems described include custom microfluidic chips and custom (benchtop) chip control devices for automated assays steps. Also included are partition formats for digital assays and lateral flow biosensors as a readout modality. CRISPR-based, microfluidics-driven assays offer highly specific detection and are compatible with parallel, combinatorial implementation. They are highly reconfigurable, and assays are compatible with isothermal and even room temperature operation. A major drawback of these assays is the fact that reports of kinetic rates of these enzymes have been highly inconsistent (many demonstrably erroneous), and the low kinetic rate activity of these enzymes limits achievable sensitivity without pre-amplification. Further, the current state-of-the-art of CRISPR assays is such that nearly all systems rely on off-chip assays steps, particularly off-chip sample preparation.

Microfluidic mixers offer new possibilities for the study of fast reaction kinetics down to the microsecond time scale, and methods such as soft X-ray absorption spectroscopy are powerful analysis techniques. These systems impose challenging constraints on mixing time scales, sample volume, detection region size and component materials. The current work presents a novel micromixer and jet device which aims to address these limitations. The system uses a so-called ‘theta’ mixer consisting of two sintered and fused glass capillaries. Sample and carrier fluids are injected separately into the inlets of the adjacent capillaries. At the downstream end, the two streams exit two micron-scale adjoining nozzles and form a single free-standing jet. The flow-rate difference between the two streams results in the rapid acceleration and lamination of the sample stream. This creates a small transverse dimension and induces diffusive mixing of the sample and carrier stream solutions within a time scale of 0.9 microseconds. The reaction occurs at or very near a free surface so that reactants and products are more directly accessible to interrogation using soft X-ray. We use a simple diffusion model and quantitative measurements of fluorescence quenching (of fluorescein with potassium iodide) to characterize the mixing dynamics across flow-rate ratios.