Microfluidics: reframing biological enquiry.

The underlying physical properties of microfluidic tools have led to new biological insights through the development of microsystems that can manipulate, mimic and measure biology at a resolution that has not been possible with macroscale tools. Microsystems readily handle sub-microlitre volumes, precisely route predictable laminar fluid flows and match both perturbations and measurements to the length scales and timescales of biological systems. The advent of fabrication techniques that do not require highly specialized engineering facilities is fuelling the broad dissemination of microfluidic systems and their adaptation to specific biological questions. We describe how our understanding of molecular and cell biology is being and will continue to be advanced by precision microfluidic approaches and posit that microfluidic tools - in conjunction with advanced imaging, bioinformatics and molecular biology approaches - will transform biology into a precision science.

Reducing Cathodic Drift during Isoelectric Focusing Using Microscale Immobilized pH Gradient Gels.

Microfluidic analytical tools play an important role in miniaturizing targeted proteomic assays for improved detection sensitivity, throughput, and automation. Microfluidic isoelectric focusing (IEF) can resolve proteoforms in lysate from low-to-single cell numbers. However, IEF assays often use carrier ampholytes (CAs) to establish a pH gradient for protein separation, presenting limitations like pH instability in the form of cathodic drift (migration of focused proteins toward the cathode). Immobilized pH gradient (IPG) gels reduce cathodic drift by covalently immobilizing the pH buffering components to a matrix. To our knowledge, efforts to implement IPG gels at the microscale have been limited to glass microdevices. To adapt IEF using IPGs to widely used microfluidic device materials, we introduce a polydimethylsiloxane (PDMS)-based microfluidic device and compare the microscale pH gradient stability of IEF established with IPGs, CAs, and a hybrid formulation of IPG gels and CAs (mixed-bed IEF). The PDMS-based IPG microfluidic device (µIPG) resolved analytes differing by 0.1 isoelectric point within a 3.5 mm separation lane over a 20 min focusing duration. During the 20 min duration, we observed markedly different cathodic drift velocities among the three formulations: 60.1 µm/min in CA-IEF, 2.5 µm/min in IPG-IEF (∼24-fold reduction versus CA-IEF), and 1.4 µm/min in mixed-bed IEF (∼43-fold reduction versus CA-IEF). Lastly, mixed-bed IEF in a PDMS device resolved green fluorescent protein (GFP) proteoforms from GFP-expressing human breast cancer cell lysate, thus establishing stability in lysate from complex biospecimens. µIPG is a promising and stable technique for studying proteoforms from small volumes.

New Views of Old Proteins: Clarifying the Enigmatic Proteome.

All human diseases involve proteins, yet our current tools to characterize and quantify them are limited. To better elucidate proteins across space, time, and molecular composition, we provide a >10 years of projection for technologies to meet the challenges that protein biology presents. With a broad perspective, we discuss grand opportunities to transition the science of proteomics into a more propulsive enterprise. Extrapolating recent trends, we describe a next generation of approaches to define, quantify, and visualize the multiple dimensions of the proteome, thereby transforming our understanding and interactions with human disease in the coming decade.

Electrotransfer of Immunoprobes through Thin-Layer Polyacrylamide Gels.

Hydrogels are important structural and operative components of microfluidic systems, finding diverse utility in biological sample preparation and interrogation. One inherent challenge for integrating hydrogels into microfluidic tools is thermodynamic molecular partitioning, which reduces the in-gel concentration of molecular solutes (e.g., biomolecular regents), as compared to the solute concentration in an applied solution. Consequently, biomolecular reagent access to in-gel scaffolded biological samples (e.g., encapsulated cells, microbial cultures, target analytes) is adversely impacted in hydrogels. Further, biomolecular reagents are typically introduced to the hydrogel via diffusion. This passive process requires long incubation periods compared to active biomolecular delivery techniques. Electrotransfer is an active technique used in Western blots and other gel-based immunoassays that overcomes limitations of size exclusion (increasing the total probe mass delivered into gel) and expedites probe delivery, even in millimeter-thick slab gels. While compatible with conventional slab gels, electrotransfer has not been adapted to thin gels (50-250 µm thick), which are of great interest as components of open microfluidic devices (vs enclosed microchannel-based devices). Mechanically delicate, thin gels are often mounted on rigid support substrates (glass, plastic) that are electrically insulating. Consequently, to adapt electrotransfer to thin-gel devices, we replace rigid insulating support substrates with novel, mechanically robust, yet electrically conductive nanoporous membranes. We describe grafting nanoporous membranes to thin-polyacrylamide-gel layers via silanization, characterize the electrical conductivity of silane-treated nanoporous membranes, and report the dependence of in-gel immunoprobe concentration on transfer duration for passive diffusion and active electrotransfer. Alternative microdevice component layers─including the mechanically robust, electrically conductive nanoporous membranes reported here─provide new functionality for integration into an increasing array of open microfluidic systems.

Multiplexed Ion Beam Imaging Readout of Single-Cell Immunoblotting.

Improvements in single-cell protein analysis are required to study the cell-to-cell variation inherent to diseases, including cancer. Single-cell immunoblotting (scIB) offers proteoform detection specificity, but often relies on fluorescence-based readout and is therefore limited in multiplexing capability. Among rising multiplexed imaging methods is multiplexed ion beam imaging by time-of-flight (MIBI-TOF), a mass spectrometry imaging technology. MIBI-TOF employs metal-tagged antibodies that do not suffer from spectral overlap to the same degree as fluorophore-tagged antibodies. We report for the first-time MIBI-TOF of single-cell immunoblotting (scIB-MIBI-TOF). The scIB assay subjects single-cell lysate to protein immunoblotting on a microscale device consisting of a 50- to 75-µm thick hydrated polyacrylamide (PA) gel matrix for protein immobilization prior to in-gel immunoprobing. We confirm antibody-protein binding in the PA gel with indirect fluorescence readout of metal-tagged antibodies. Since MIBI-TOF is a layer-by-layer imaging technique, and our protein target is immobilized within a 3D PA gel layer, we characterize the protein distribution throughout the PA gel depth by fluorescence confocal microscopy and confirm that the highest signal-to-noise ratio is achieved by imaging the entirety of the PA gel depth. Accordingly, we report the required MIBI-TOF ion dose strength needed to image varying PA gel depths. Lastly, by imaging ∼42% of PA gel depth with MIBI-TOF, we detect two isoelectrically separated TurboGFP (tGFP) proteoforms from individual glioblastoma cells, demonstrating that highly multiplexed mass spectrometry-based readout is compatible with scIB.

Segmentation-based analysis of single-cell immunoblots.

From genomics to transcriptomics to proteomics, microfluidic tools underpin recent advances in single-cell biology. Detection of specific proteoforms-with single-cell resolution-presents challenges in detection specificity and sensitivity. Miniaturization of protein immunoblots to single-cell resolution mitigates these challenges. For example, in microfluidic western blotting, protein targets are separated by electrophoresis and subsequently detected using fluorescently labeled antibody probes. To quantify the expression level of each protein target, the fluorescent protein bands are fit to Gaussians; yet, this method is difficult to use with noisy, low-abundance, or low-SNR protein bands, and with significant band skew or dispersion. In this study, we investigate segmentation-based approaches to robustly quantify protein bands from single-cell protein immunoblots. As compared to a Gaussian fitting pipeline, the segmentation pipeline detects >1.5× more protein bands for downstream quantification as well as more of the low-abundance protein bands (i.e., with SNR ∼3). Utilizing deep learning-based segmentation approaches increases the recovery of low-SNR protein bands by an additional 50%. However, we find that segmentation-based approaches are less robust at quantifying poorly resolved protein bands (separation resolution, Rs < 0.6). With burgeoning needs for more single-cell protein analysis tools, we see microfluidic separations as benefitting substantially from segmentation-based analysis approaches.

Comparison of photoactivatable crosslinkers for in-gel immunoassays.

While fluorescence readout is a key detection modality for hydrogel-based immunoassays, background fluorescence due to autofluorescence or non-specific antibody interactions impairs the lower limit of detection of fluorescence immunoassays. Chemical modifications to the hydrogel structure impact autofluorescence and non-specific interactions. Benzophenone is a common photoactivatable molecule, and benzophenone methacrylamide (BPMA) has been used for cross-linking protein in polyacrylamide (PA) hydrogels. However, previous studies have suggested that the aromatic structure of benzophenone can contribute to increased autofluorescence and non-specific hydrophobic interactions with unbound fluorescent probes. Here, we synthesize diazirine methacrylamide (DZMA) as an alternative photoactivatable molecule to crosslink into PA hydrogels for in-gel protein capture for in-gel immunoassays. We hypothesize that the less hydrophobic structure of diazirine (based on previously reported predicted and experimental log P values) exhibits both reduced autofluorescence and non-specific hydrophobic interactions. We find that while equal concentrations of DZMA and BPMA result in lower protein target photocapture in the diazirine configuration, increasing the DZMA concentration up to 12 mM improves in-gel protein capture to be on par with previously reported and characterized 3 mM BPMA hydrogels. Furthermore, despite the higher concentration of diazirine, we observe negligible autofluorescence signal and a 50% reduction in immunoassay fluorescence background signal in diazirine gels compared to BPMA gels resulting in comparable signal-to-noise ratios (SNR) of the probed protein target. Finally, we test the utility of DZMA for single-cell immunoblotting in an open microfluidic device and find that protein migrates ∼1.3× faster in DZMA hydrogels than in BPMA hydrogels. However, in DZMA hydrogels we detect only 15% of the protein signal compared to BPMA hydrogels suggesting that the diazirine chemistry results in greater protein losses following electrophoretic separations. We establish that while diazirine has lower background fluorescence signal, which may potentially improve immunoassay performance, the lower capture efficiency of diazirine reduces its utility in open microfluidic systems susceptible to sample losses.

Best Practices for Germicidal Ultraviolet-C Dose Measurement for N95 Respirator Decontamination.

Ultraviolet-C (UV-C) decontamination holds promise in combating the coronavirus disease 2019 pandemic, particularly with its potential to mitigate the N95 respirator shortage. Safe, effective, and reproducible decontamination depends critically on UV-C dose, yet dose is frequently measured and reported incorrectly, which results in misleading and potentially harmful protocols. Understanding best practices in UV-C dose measurement for N95 respirator decontamination is essential to the safety of medical professionals, researchers, and the public. Here, we outline the fundamental optical principles governing UV-C irradiation and detection, as well as the key metrics of UV-C wavelength and dose. In particular, we discuss the technical and regulatory distinctions between UV-C N95 respirator decontamination and other applications of germicidal UV-C, and we highlight the unique considerations required for UV-C N95 respirator decontamination. Together, this discussion will inform best practices for UV-C dose measurement for N95 respirator decontamination during crisis-capacity conditions.

In Situ Measurement of Thermodynamic Partitioning in Open Hydrogels.

Thermodynamic partitioning dictates solute loading and release from a hydrogel. Design of drug delivery vehicles, cell and tissue matrices, and immunoassay scaffolds that utilize hydrogel materials is informed by an understanding of the thermodynamic partitioning properties of those hydrogels. We develop aberration-compensated laser scanning confocal microscopy (AC-LSCM), a technique that can be applied to all fluorescence microscopy-based equilibrium partition coefficient measurements where the fluorescence is uniformly distributed in the reference material (e.g., many solutes in thermodynamic equilibrium). In this paper, we use AC-LSCM to measure spatially resolved in situ equilibrium partition coefficients of various fluorescently labeled solutes in single-layer and multilayer open hydrogels. In considering a dynamic material, we scrutinize solute interactions with a UV photoactive polyacrylamide gel that incorporates a benzophenone methacrylamide backbone. We observed strong agreement with an adjusted version of Ogston's ideal size-exclusion model for spatially resolved in situ equilibrium partition coefficients across a wide range of polyacrylamide hydrogel densities (R2 = 0.98). Partition coefficients of solutes differing in hydrodynamic radius were consistent with size-based theory in the photoactive hydrogels, but exceed those in unmodified polyacrylamide gels. This observation suggests a deviation from the size-exclusion model and a shift in the thermodynamic equilibrium state of the solutes toward the gel phase. AC-LSCM also resolves differential partitioning behavior of the model solute in two-layer gels, providing insight into the transport phenomena governing the partitioning in multilaminate gel structures. Furthermore, AC-LSCM identifies and quantifies depth-dependent axial aberrations that could confound quantitation, highlighting the need for the "aberration compensated" aspect of AC-LSCM.

Laterally Aggregated Polyacrylamide Gels for Immunoprobed Isoelectric Focusing.

Immunoprobed isoelectric focusing (IEF) resolves proteins based on differences in isoelectric point (pI) and then identifies protein targets through immunoprobing of IEF-separated proteins that have been immobilized onto a gel scaffold. During the IEF stage, the gel functions as an anti-convective medium and not as a molecular sieving matrix. During the immunoprobing stage, the gel acts as an immobilization scaffold for IEF-focused proteins via photoactive moieties. Here, we characterized the effect of gel pore size on IEF separation and in-gel immunoassay performance. We modulated polyacrylamide (PA) gel pore size via lateral chain aggregation initiated by PEG monomers. During IEF, the 2% PEG highly porous PA gel formulation offered higher resolution (minimum pI difference ∼0.07 ± 0.02) than unmodified 6%T, 3.3%C (benchmark) and 6%T, 8%C (negative control) PA gels. The highly porous gels supported a pH gradient with slope and linearity comparable to benchmark gels. The partition coefficient for antibodies into the highly porous gels (K = 0.35 ± 0.02) was greater than the benchmark (3×) and negative control (1.75×) gels. The highly porous gels also had lower immunoassay background signal than the benchmark (2×) and negative control (3×) gels. Taken together, lateral aggregation creates PA gels that are suitable for both IEF and subsequent in-gel immunoprobing by mitigating immunoprobe exclusion from the gels while facilitating removal of unbound immunoprobe.

Reversible Functionalization of Clickable Polyacrylamide Gels with Protein and Graft Copolymers.

Modular strategies to fabricate gels with tailorable chemical functionalities are relevant to applications spanning from biomedicine to analytical chemistry. Here, the properties of clickable poly(acrylamide-co-propargyl acrylate) (pAPA) hydrogels are modified via sequential in-gel copper-catalyzed azide-alkyne cycloaddition (CuAAC) reactions. Under optimized conditions, each in-gel CuAAC reaction proceeds with rate constants of ~0.003 s-1, ensuring uniform modifications for gels < 200 µm thick. Using the modular functionalization approach and a cleavable disulfide linker, pAPA gels were modified with benzophenone and acrylate groups. Benzophenone groups allow gel functionalization with unmodified proteins using photoactivation. Acrylate groups enabled copolymer grafting onto the gels. To release the functionalized unit, pAPA gels were treated with disulfide reducing agents, which triggered ~50 % release of immobilized protein and grafted copolymers. The molecular mass of grafted copolymers (~6.2 kDa) was estimated by monitoring the release process, expanding the tools available to characterize copolymers grafted onto hydrogels. Investigation of the efficiency of in-gel CuAAC reactions revealed limitations of the sequential modification approach, as well as guidelines to convert a pAPA gel with a single functional group into a gel with three distinct functionalities. Taken together, we see this modular framework to engineer multifunctional hydrogels as benefiting applications of hydrogels in drug delivery, tissue engineering, and separation science.

Ferguson analysis of protein electromigration during single-cell electrophoresis in an open microfluidic device.

In an open microfluidic device, we investigate protein polyacrylamide gel electrophoresis (PAGE) separation performance on single-cell lysate. Single-cell protein electrophoresis is performed in a thin layer of polyacrylamide (PA) gel into which microwells are molded. Individual cells are isolated in these open microwells, then lysed on-chip with a dual lysis and electrophoresis sodium dodecyl sulfate (SDS) buffer. We scrutinize the effect of sieving gel composition on electromigration of protein targets, using a wide range of cellular protein standards (36 kDa to 289 kDa). We find that as PA concentration increases, protein electromigration deviates from the empirical log-linear relationship predicted between migration distance and molecular mass. We perform Ferguson analysis to calculate retardation coefficients and free solution mobilities of nine cellular protein standards and observe that the largest-molecular-mass protein, mTOR (289 kDa), does not behave as predicted by established linear-fit models for SDS-denatured proteins, indicating that mTOR is beyond the linear range of this assay. Lastly, we performed in-gel immunoprobing on the single-cell electrophoretic separations and observed that smaller pore-size gels (higher gel concentration) reduce protein diffusion out of the gel, which does not notably impact the measured immunoprobed protein expression. Compared to larger pore-size gels, the smaller pore-size gels lead to higher local concentrations of the target protein in each protein band, resulting in an increase in the signal-to-noise ratio (SNR) for each protein. Understanding the separation and immunoprobing performance at different gel concentrations improves assay design and optimization for target proteins.

Single-cell mobility shift electrophoresis reports protein localization to the cell membrane.

While profiling of cell surface receptors grants valuable insight on cell phenotype, surface receptors alone cannot fully describe activated downstream signaling pathways, detect internalized receptor activity, or indicate constitutively active signaling in subcellular compartments. To measure surface-bound and intracellular targets in the same cell, we introduce a tandem single-cell assay that combines immunofluorescence of surface-bound epithelial cellular adhesion molecule (EpCAM) with subsequent protein polyacrylamide gel electrophoresis (PAGE) of unfixed MCF7 breast cancer cells. After surface staining and cell lysis, surface EpCAM is analyzed by single-cell PAGE, concurrent with immunoprobing of intracellular targets. Consequently, the single-cell electrophoresis step reports localization of both surface and intracellular targets. Unbound intracellular EpCAM is readily resolved from surface EpCAM immunocomplex owing to a ∼30% mobility shift. Flow cytometry and immunofluorescence are in concordance with single-cell PAGE. Lastly, we challenged the stability of the EpCAM immunocomplexes by varying ionic and non-ionic component concentrations in the lysis buffer, the lysis time, and electrophoresis duration. As expected, the harsher conditions proved most disruptive to the immunocomplexes. The compatibility of live-cell immunostaining with single-cell PAGE eliminates the need to perform single-cell imaging by condensing read-out of both surface-bound proteins (as low mobility immune complexes) and intracellular targets to a single immunoblot, thus linking cell type and state.

In Situ Single-Cell Western Blot on Adherent Cell Culture.

Integrating 2D culture of adherent mammalian cells with single-cell western blotting (in situ scWB) uses microfluidic design to eliminate the requirement for trypsin release of cells to suspension, prior to single-cell isolation and protein analysis. To assay HeLa cells from an attached starting state, we culture adherent cells in fibronectin-functionalized microwells formed in a thin layer of polyacrylamide gel. To integrate the culture, lysis, and assay workflow, we introduce a one-step copolymerization process that creates protein-decorated microwells. After single-cell culture, we lyse each cell in the microwell and perform western blotting on each resultant lysate. We observe cell spreading after overnight microwell-based culture. scWB reports increased phosphorylation of MAP kinases (ERK1/2, p38) under hypertonic conditions. We validate the in situ scWB with slab-gel western blot, while revealing cell-to-cell heterogeneity in stress responses.

Controlling Dispersion during Single-Cell Polyacrylamide-Gel Electrophoresis in Open Microfluidic Devices.

New tools for measuring protein expression in individual cells complement single-cell genomics and transcriptomics. To characterize a population of individual mammalian cells, hundreds to thousands of microwells are arrayed on a polyacrylamide-gel-coated glass microscope slide. In this "open" fluidic device format, we explore the feasibility of mitigating diffusional losses during lysis and polyacrylamide-gel electrophoresis (PAGE) through spatial control of the pore-size of the gel layer. To reduce in-plane diffusion-driven dilution of each single-cell lysate during in-microwell chemical lysis, we photopattern and characterize microwells with small-pore-size sidewalls ringing the microwell except at the injection region. To reduce out-of-plane-diffusion-driven-dilution-caused signal loss during both lysis and single-cell PAGE, we scrutinize a selectively permeable agarose lid layer. To reduce injection dispersion, we photopattern and study a stacking-gel feature at the head of each <1 mm separation axis. Lastly, we explore a semienclosed device design that reduces the cross-sectional area of the chip, thus reducing Joule-heating-induced dispersion during single-cell PAGE. As a result, we observed a 3-fold increase in separation resolution during a 30 s separation and a >2-fold enhancement of the signal-to-noise ratio. We present well-integrated strategies for enhancing overall single-cell-PAGE performance.

Microparticle Delivery of Protein Markers for Single-Cell Western Blotting from Microwells.

Immunoblotting confers protein identification specificity beyond that of immunoassays by prepending protein electrophoresis (sizing) to immunoprobing. To accurately size protein targets, sample analysis includes concurrent analysis of protein markers with known molecular masses. To incorporate protein markers in single-cell western blotting, microwells are used to isolate individual cells and protein marker-coated microparticles. A magnetic field directs protein-coated microparticles to >75% of microwells, so as to 1) deliver a quantum of protein marker to each cell-laden microwell and 2) synchronize protein marker solubilization with cell lysis. Nickel-coated microparticles are designed, fabricated, and characterized, each conjugated with a mixture of histidine-tagged proteins (42.3-100 kDa). Imidazole in the cell lysis buffer solubilizes protein markers during a 30 s cell lysis step, with an observed protein marker release half-life of 4.46 s. Across hundreds of individual microwells and different microdevices, robust log-linear regression fits (R2 > 0.97) of protein molecular mass and electrophoretic mobility are observed. The protein marker and microparticle system is applied to determine the molecular masses of five endogenous proteins in breast cancer cells (GAPDH, ß-TUB, CK8, STAT3, ER-α), with <20% mass error. Microparticle-delivered protein standards underpin robust, reproducible electrophoretic cytometry that complements single-cell genomics and transcriptomics.

Arrayed isoelectric focusing using photopatterned multi-domain hydrogels.

Isoelectric focusing (IEF) is a powerful separation method, useful for resolving subtle changes in the isoelectric point of unlabeled proteins. While microfluidic IEF has reduced the separation times from hours in traditional benchtop IEF to minutes, the enclosed devices hinder post-separation access to the sample for downstream analysis. The two-layer open IEF device presented here comprises a photopatterned hydrogel lid layer containing the chemistries required for IEF and a thin polyacrylamide bottom layer in which the analytes are separated. The open IEF device produces comparable minimum resolvable difference in isoelectric point and gradient stability to enclosed microfluidic devices while providing post-separation sample access by simple removal of the lid layer. Further, using simulations, we determine that the material properties and the length of the separation lanes are the primary factors that affect the electric field magnitude in the separation region. Finally, we demonstrate self-indexed photomasks for alignment-free fabrication of multi-domain hydrogels. We leverage this approach to generate arrayed pH gradients with a total of 80 concurrent separation lanes, which to our knowledge is the first demonstration of multiple IEF separations in series addressed by a single pair of electrodes.

Rapid Capture and Release of Nucleic Acids through a Reversible Photo-Cycloaddition Reaction in a Psoralen-Functionalized Hydrogel.

Reversible immobilization of DNA and RNA is of great interest to researchers who seek to manipulate DNA or RNA in applications such as microarrays, DNA hydrogels, and gene therapeutics. However, there is no existing system that can rapidly capture and release intact nucleic acids. To meet this unmet need, we developed a functional hydrogel for rapid DNA/RNA capture and release based on the reversible photo-cycloaddition of psoralen and pyrimidines. The functional hydrogel can be easily fabricated through copolymerization of acrylamide with the synthesized allylated psoralen. The psoralen-functionalized hydrogel exhibits effective capture and release of nucleic acids spanning a wide range of lengths in a rapid fashion; over 90 % of the capture process is completed within 1 min, and circa 100 % of the release process is completed within 2 min. We observe no deleterious effects on the hybridization to the captured targets.

Kinetic Analysis of Enzymes Immobilized in Porous Film Arrays.

Measuring the catalytic activity of immobilized enzymes underpins development of biosensing, bioprocessing, and analytical chemistry tools. To expand the range of approaches available for measuring enzymatic activity, we report on a technique to probe activity of enzymes immobilized in porous materials in the absence of confounding mass transport artifacts. We measured reaction kinetics of calf intestinal alkaline phosphatase (CIAP) immobilized in benzophenone-modified polyacrylamide (BPMA-PAAm) gel films housed in an array of fluidically isolated chambers. To ensure kinetics measurements are not confounded by mass transport limitations, we employed Weisz's modulus (Φ), which compares observed enzyme-catalyzed reaction rates to characteristic substrate diffusion times. We characterized activity of CIAP immobilized in BPMA-PAAm gels in a reaction-limited regime (Φ â‰ª 0.15 for all measurements), allowing us to isolate the effect of immobilization on enzymatic activity. Immobilization of CIAP in BPMA-PAAm gels produced a ∼2× loss in apparent enzyme-substrate affinity (Km) and ∼200× decrease in intrinsic catalytic activity (kcat) relative to in-solution measurements. As estimating Km and kcat requires multiple steps of data manipulation, we developed a computational approach (bootstrapping) to propagate uncertainty in calibration data through all data manipulation steps. Numerical simulation revealed that calibration error is only negligible when the normalized root-mean-squared error (NRMSE) in the calibration falls below 0.05%. Importantly, bootstrapping is independent of the mathematical model, and thus generalizable beyond enzyme kinetics studies. Furthermore, the measurement tool presented can be readily adapted to study other porous immobilization supports, facilitating rational design (immobilization method, geometry, enzyme loading) of immobilized-enzyme devices.