Probing multivalent particle–surface interactions using a quartz crystal resonator

The rise in market-approved cellular therapies demands for advancements in process analytical technology (PAT) capable of fulfilling the requirements of this new industry. Unlike conventional biopharmaceuticals, cell-based therapies (CBT) are complex “live” products, with a high degree of inherent biological variability. This exacerbates the need for in-process monitoring and control of critical product attributes, in order to guarantee safety, efficacious and continuous supply of this CBT. There are therefore mutual industrial and regulatory motivations for high throughput, non-invasive and non-destructive sensors, amenable to integration in an enclosed automated cell culture system. While a plethora of analytical methods is available for direct characterization of cellular parameters, only a few satisfy the requirements for online quality monitoring of industrial-scale bioprocesses. [Continues.

Supplementary information files for A quartz crystal resonator for cellular phenotyping

Supplementary files for article A quartz crystal resonator for cellular phenotyping. Cell therapy manufacturing is limited by lack of online tools capable of realtime in-process monitoring, particularly of simultaneous changes in multiple orthogonal (mutually independent) parameters. Here, we studied changes in CD36 expression, number density and size (area) of erythroblasts through different stages of erythropoiesis in vitro using a quartz crystal resonator (QCR), integrated with a microscope, and flow cytometry in parallel. An analytical model was developed extending the Kanazawa-Gordon theory. Based on this model, independent correlations were established between changes in each QCR parameter, dissipation (∆Γ) and resonance frequency (〖-Δf〗_0), and CD36 expression (from flow cytometry) and cell area (from microscope). The correlation functions were used to derive an acoustic signature (-∆Γ/〖Δf〗_0) of the differentiation process that uniquely mapped the relative changes in CD36 expression and late-stage enucleation-related deviations. A method to quantify relative changes in cell area purely from the acoustic parameters was also proposed. This work demonstrated for the first time the potential of an electromechanical tool for online monitoring of concurrently varying orthogonal phenotypic parameters in cell therapy manufacturing.</div

A quartz crystal resonator for cellular phenotyping

Cell therapy manufacturing is limited by lack of online tools capable of realtime in-process monitoring, particularly of simultaneous changes in multiple orthogonal (mutually independent) parameters. Here, we studied changes in CD36 expression, number density and size (area) of erythroblasts through different stages of erythropoiesis in vitro using a quartz crystal resonator (QCR), integrated with a microscope, and flow cytometry in parallel. An analytical model was developed extending the Kanazawa-Gordon theory. Based on this model, independent correlations were established between changes in each QCR parameter, dissipation (∆Γ) and resonance frequency (〖-Δf〗_0), and CD36 expression (from flow cytometry) and cell area (from microscope). The correlation functions were used to derive an acoustic signature (-∆Γ/〖Δf〗_0) of the differentiation process that uniquely mapped the relative changes in CD36 expression and late-stage enucleation-related deviations. A method to quantify relative changes in cell area purely from the acoustic parameters was also proposed. This work demonstrated for the first time the potential of an electromechanical tool for online monitoring of concurrently varying orthogonal phenotypic parameters in cell therapy manufacturing

Supplementary Information files for "Characterisation of particle-surface interactions via anharmonic acoustic transduction"

These are the SI files for the article "Characterisation of particle-surface interactions via anharmonic acoustic transduction".Abstract:Most transduction methods for measuring particle-surface interactions are unable to differentiate the strength of interaction and largely reliant on extensive washing to reduce the ubiquitous non-specific background. Label-based methods, in particular, are limited in wide applicability due to their inherent operational complexity. On the other hand, label-free force-spectroscopic methods that can differentiate particle-surface interaction strength are skill-demanding and time-consuming. Here, we present a label-free anharmonic (nonlinear) acoustic transduction method employing the quartz crystal resonator that reads out ligand-receptor binding based on the interaction strength. We show that while stronger specific interactions are transduced more strongly, and in linear proportionality to the ligand concentration on microparticles, non-specific interactions are significantly attenuated. This allows ligand quantification with high specificity and sensitivity in realtime under flow without separate washing steps. Constructing an analytical model of a quartz resonator, we can relate the number and type (specific vs. non-specific) of ligand-receptor interactions with the change in characteristic nonlinearity coefficient of the resonator. The entirely-electronic and microfluidic-integrable transduction method could potentially allow a simple, fast and reliable way for characterising particle-surface interactions with economy of scale.</div

Supplementary information files for article: Direct detection of whole bacteria using a nonlinear acoustic resonator

Direct detection of whole vegetative bacteria was investigated employing a quartz crystal resonator (QCR) in its nonlinear regime. Escherichia coli (E. coli) in buffer solution under flow was captured on a QCR in a microfluidic cell using a whole-cell anti-E.coli aptamer. The nonlinear distortion in QCR response due to the ‘pull’ from surface-bound bacteria was measured in realtime as the change in third Fourier harmonic (3f) current and compared with shifts in the traditional acoustic parameters of resonance frequency and dissipation. The change in 3f current showed superior quantitative correlation with E. coli concentrations (105-108 cfu/mL) and at least an order of magnitude better sensitivity than shifts in the traditional acoustic parameters. Most interestingly, underpinned by the strength of bacteria-QCR pull, the nonlinear acoustic principle demonstrated a unique specificity in transduction, even in a mixed sample with another gram-negative bacteria, that can supplement the specificity of the bioreceptors. An analytical expression was derived to quantitatively relate the competing influence of shifts in dissipation and nonlinearity coefficient of the QCR on the change in 3f current. This study demonstrates the potential for reliable direct readout of bioreceptor-mediated binding of whole vegetative bacteria from complex samples using a nonlinear acoustic resonator

Direct detection of whole bacteria using a nonlinear acoustic resonator

Direct detection of whole vegetative bacteria was investigated employing a quartz crystal resonator (QCR) in its nonlinear regime. Escherichia coli (E. coli) in buffer solution under flow was captured on a QCR in a microfluidic cell using a whole-cell anti-E.coli aptamer. The nonlinear distortion in QCR response due to the ‘pull’ from surface-bound bacteria was measured in realtime as the change in third Fourier harmonic (3f) current and compared with shifts in the traditional acoustic parameters of resonance frequency and dissipation. The change in 3f current showed superior quantitative correlation with E. coli concentrations (105-108 cfu/mL) and at least an order of magnitude better sensitivity than shifts in the traditional acoustic parameters. Most interestingly, underpinned by the strength of bacteria-QCR pull, the nonlinear acoustic principle demonstrated a unique specificity in transduction, even in a mixed sample with another gram-negative bacteria, that can supplement the specificity of the bioreceptors. An analytical expression was derived to quantitatively relate the competing influence of shifts in dissipation and nonlinearity coefficient of the QCR on the change in 3f current. This study demonstrates the potential for reliable direct readout of bioreceptor-mediated binding of whole vegetative bacteria from complex samples using a nonlinear acoustic resonator.</p