Response to the comments on "Cellular aggregation dictates universal spreading behaviour of a whole-blood drop on a paper strip".

In 2023, we published a research article in the Journal of Colloidal and Interface Science, based on our experimental findings and substantiating scaling arguments leading to a simple theoretical insight on the effect of red blood cell (RBC) aggregation on the wicking behaviour of a finite volume of blood as it navigates through the porous passages of a paper matrix (Laha et al., 2023). Of late, we received comments from Li (2024), which offered certain suggestions regarding the possible improvement of the capillary bundle model as considered in our article for analyzing the transport of blood through the paper pores. Herein, we provide a detailed discussion on each of the points raised by Li (2024) and rationalize our views in further details in addition to the contents already provided in our concerned article.

Microfluidics-Based Blood Typing Devices: An In-Depth Overview.

Identification of correct blood types holds paramount importance in understanding the pathophysiological parameters of patients, therapeutic interventions, and blood transfusion. Considering the wide applications of blood typing, the requirement of centralized laboratory facilities is not well suited on many occasions. In this context, there has been a significant development of such blood typing devices on different microfluidic platforms. The advantages of these microfluidic devices offer easy, rapid test protocols, which could potentially be adapted in resource-limited settings and thereby can truly lead to the decentralization of testing facilities. The advantages of pump-free liquid transport (i.e., low power consumption) and biodegradability of paper substrates (e.g., reduction in medical wastes) make it a more preferred platform in comparison to other microfluidic devices. However, these devices are often coupled with some inherent challenges, which limit their potential to be used on a mass commercial scale. In this context, our Review offers a succinct summary of the recent development, especially to understand the importance of underlying facets for long-term sustainability. Our Review also delineates the role of integration with digital technologies to minimize errors in interpreting the readouts.

Real-time detection of plasma ferritin by electrochemical biosensor developed for biomedical analysis.

Rapid quantification of plasma ferritin levels holds utmost importance for the effective management of different chronic illnesses. We report the development of a novel biosensor for quantitative and selective detection of ferritin from a drop of blood plasma. Developed electrochemical biosensing platform contains a semiconductor nano-structured decorated screen-printed electrode (SND-SPE). The hydrothermally synthesized ZnO-Mn3O4 nanocomposite which has been coated on the electrode surfaces, imparts the specificity in ferritin diagnostics. Cyclic voltametric (CV) measurements with blood plasma shows a prominent reduction peak of ∼ - 0.76 V for specific ferritin reduction. The amperometric sensor shows a known concentration of 0.3 µg/dl ferritin-containing plasma generates 15 µA of current for single-time use. The efficacy of the device is evaluated by detecting ferritin in human plasma samples. The limit of detection and response time of the developed sensor are 0.04 µg/dl and 0.1 s respectively. The layer of ZnO-Mn3O4 nanocomposite has played as an excellent catalyst during the specific reduction of Fe3+ ion and the merits of the device in terms of high robustness, ultrafast detection, highly stable, low-cost, and a biodegradable sensor, make it attractive for the deployment in point-of-care settings.

Cellular aggregation dictates universal spreading behaviour of a whole-blood drop on a paper strip.

HYPOTHESIS: The complex spreading dynamics of blood on paper matrix is likely to be quantitatively altered with variations in the fractional occupancy of red blood cells in the whole blood (haematocrit). Here, we presented an apparently surprising observation that a finite volume blood drop undergoes a universal time-dependent spreading on a filter paper strip that is virtually invariant with its hematocrit level within physiologically healthy regime, though distinctively distinguishable from the spreading laws of blood plasma and water. EXPERIMENTS: Our hypothesis was ascertained by performing controlled wicking experiments on filter papers of different grades. Spreading of human blood samples of different haematocrit levels ranging between 15% and 51% and the plasma separated from therein were traced by combined high-speed imaging and microscopy. These experiments were complemented with a semi-analytical theory to decipher the key physics of interest. RESULTS: Our results unveiled the exclusive influence of the obstructing cellular aggregates in the randomly distributed hierarchically structured porous pathways and deciphered the role of the networked structures of the various plasma proteins that induced hindered diffusion. The resulting universal signatures of spontaneous dynamic spreading, delving centrally on the fractional reduction in the interlaced porous passages, provide novel design basis for paper-microfluidic kits in medical diagnostics and beyond.

Smartphone-Enabled Paper-Based Hemoglobin Sensor for Extreme Point-of-Care Diagnostics.

We report a simple, affordable (∼0.02 US $/test), rapid (within 5 min), and quantitative paper-based sensor integrated with smartphone application for on-spot detection of hemoglobin (Hgb) concentration using approximately 10 µL of finger-pricked blood. Quantitative analytical colorimetry is achieved via an Android-based application (Sens-Hb), integrating key operational steps of image acquisition, real-time analysis, and result dissemination. Further, feedback from the machine learning algorithm for adaptation of calibration data offers consistent dynamic improvement for precise predictions of the test results. Our study reveals a successful deployment of the extreme point-of-care test in rural settings where no infrastructural facilities for diagnostics are available. The Hgb test device is validated both in the controlled laboratory environment (n = 200) and on the field experiments (n = 142) executed in four different Indian villages. Validation results are well correlated with the pathological gold standard results (r = 0.9583) with high sensitivity and specificity for the healthy (n = 136) (>11 g/dL) (specificity: 97.2%), mildly anemic (n = 55) (<11 g/dL) (sensitivity: 87.5%, specificity: 100%), and severely anemic (n = 9) (<7 g/dL) (sensitivity: 100%, specificity: 100%) samples. Results from field trials reveal that only below 5% cases of the results are interpreted erroneously by classifying mildly anemic patients as healthy ones. On-field deployment has unveiled the test kit to be extremely user friendly that can be handled by minimally trained frontline workers for catering the needs of the underserved communities.

Formation of Blood Droplets: Influence of the Plasma Proteins.

Blood is a complex multiphase fluid exhibiting pronounced shear-thinning and viscoelastic behavior. By studying the formation of blood droplets through simple dripping, we observe blood-drop detachment following a neck formation and subsequent thinning until breakup, similar to that of other liquids. Our experimental findings reveal that it exhibits two distinct modes of neck evolution characteristics; one mode corresponds to incessant collapsing of the liquid neck, whereas the other mode correlates thinning of an extended long thread leading to the breakup. We show that the two modes of neck evolution closely follow the theory of pinch-off for shear-thinning and viscoelastic fluids independent of hematocrit concentration in the range of healthy individuals. Furthermore, we observe that the relaxation time scales are very similar to that of plasma; this explains the key role of plasma proteins to blood rheology. We envision that our results are likely to bear far-reaching implications in understanding the contribution of plasma proteins to the rheology of blood and theory of drop formation of complex non-Newtonian fluids.

Hydroelectric power plant on a paper strip.

We exploit the combinatorial advantage of electrokinetics and tortuosity of a cellulose-based paper network on laboratory grade filter paper for the development of a simple, inexpensive, yet extremely robust (shows constant performance for 12 days) 'paper-and-pencil'-based device for energy harvesting applications. We successfully achieve harvesting of a maximum output power of ∼640 pW in a single channel, while the same is significantly improved (by ∼100 times) with the use of a multichannel microfluidic array (maximum of up to 20 channels). Furthermore, we also provide theoretical insights into the observed phenomenon and show that the experimentally predicted trends agree well with our theoretical calculations. Thus, we envisage that such ultra-low cost devices may turn out to be extremely useful in energizing analytical microdevices in resource limited settings, for instance, in extreme point of care diagnostic applications.

Predicting Escherichia coli's chemotactic drift under exponential gradient.

Bacterial species are known to show chemotaxis, i.e., the directed motions in the presence of certain chemicals, whereas the motion is random in the absence of those chemicals. The bacteria modulate their run time to induce chemotactic drift towards the attractant chemicals and away from the repellent chemicals. However, the existing theoretical knowledge does not exhibit a proper match with experimental validation, and hence there is a need for developing alternate models and validating experimentally. In this paper a more robust theoretical model is proposed to investigate chemotactic drift of peritrichous Escherichia coli under an exponential nutrient gradient. An exponential gradient is used to understand the steady state behavior of drift because of the logarithmic functionality of the chemosensory receptors. Our theoretical estimations are validated through the experimentation and simulation results. Thus, the developed model successfully delineates the run time, run trajectory, and drift velocity as measured from the experiments.

Haemoglobin content modulated deformation dynamics of red blood cells on a compact disc.

We investigate the deformation characteristics of red blood cells (RBCs) on a rotating compact disc platform. Our study brings out the interplay between haemoglobin content and RBC deformability in a centrifugally actuated microfluidic environment. We reveal that RBC deformations follow the similar trend of principal stress distributed throughout the radial direction, rendering an insight into the mechano-physical processes involved. This study can be used as a diagnostic marker to determine haematological disorders in diseased blood samples tested on compact disc based microfluidic platforms.

Capillarity-driven blood plasma separation on paper-based devices.

We demonstrate capillarity-driven plasma separation from whole blood on simple paper-based H-channels. This methodology, unlike other reported techniques, does not necessitate elaborate and complex instrumentation, and the usage of expensive consumables. We believe that this technique will be ideally suited to be implemented in rapid and portable blood diagnostic devices designed to be operative at locations with limited resources.

Effect of hematocrit on blood dynamics on a compact disc platform.

We investigate blood flow dynamics on a rotationally actuated lab-on-a-compact disk (LOCD) platform, as a function of the hematocrit level of the blood sample. In particular, we emphasize the resultant implications on the critical fluidic parameters, such as on burst frequency and volumetric flow rate. Our results can be utilized as a characteristic guideline to predict the hematological parameters of a given small amount of blood sample from the observed flow characteristics, and can give rise to a new paradigm of medical diagnostics driven by interactions between blood rheology and rotational forces on an inexpensive platform, with minimal sample consumption.

Protein-directed synthesis of NIR-emitting, tunable HgS quantum dots and their applications in metal-ion sensing.

The development of luminescent mercury sulfide quantum dots (HgS QDs) through the bio-mineralization process has remained unexplored. Herein, a simple, two-step route for the synthesis of HgS quantum dots in bovine serum albumin (BSA) is reported. The QDs are characterized by UV-vis spectroscopy, Fourier transform infrared (FT-IR) spectroscopy, luminescence, Raman spectroscopy, transmission electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS), circular dichroism (CD), energy dispersive X-ray analysis (EDX), and picosecond-resolved optical spectroscopy. Formation of various sizes of QDs is observed by modifying the conditions suitably. The QDs also show tunable luminescence over the 680-800 nm spectral regions, with a quantum yield of 4-5%. The as-prepared QDs can serve as selective sensor materials for Hg(II) and Cu(II), based on selective luminescence quenching. The quenching mechanism is found to be based on Dexter energy transfer and photoinduced electron transfer for Hg(II) and Cu(II), respectively. The simple synthesis route of protein-capped HgS QDs would provide additional impetus to explore applications for these materials.

An improved microfluidics approach for monitoring real-time interaction profiles of ultrafast molecular recognition.

Our study illustrates the development of a microfluidics (MF) platform combining fluorescence microscopy and femtosecond/picosecond-resolved spectroscopy to investigate ultrafast chemical processes in liquid-phase diffusion-controlled reactions. By controlling the flow rates of two reactants in a specially designed MF chip, sub-100 ns time resolution for the exploration of chemical intermediates of the reaction in the MF channel has been achieved. Our system clearly rules out the possibility of formation of any intermediate reaction product in a so-called fast ionic reaction between sodium hydroxide and phenolphthalein, and reveals a microsecond time scale associated with the formation of the reaction product. We have also used the developed system for the investigation of intermediate states in the molecular recognition of various macromolecular self-assemblies (micelles) and genomic DNA by small organic ligands (Hoechst 33258 and ethidium bromide). We propose our MF-based system to be an alternative to the existing millisecond-resolved "stopped-flow" technique for a broad range of time-resolved (sub-100 ns to minutes) experiments on complex chemical∕biological systems.