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Drying of biologically-relevant sessile droplets, including passive systems (like DNA and proteins), as well as active microbial systems comprising bacteria and algae, have garnered considerable attention over the last decades. Distinct morphological patterns emerge when bio-colloids undergo drying, with significant potential in a range of biomedical applications, spanning bio-sensing, medical diagnostics, drug delivery, and antimicrobial resistance. This review presents a comprehensive overview of bio-colloidal droplets drying on solid substrates, focusing on the experimental progress during the last ten years. We provide a summary of the relevant properties of bio-colloids and link their composition (constituent particles, solvent, and concentrations) to the patterns emerging due to drying. We examined the drying patterns generated by passive bio-colloids (DNA, globular, fibrous, and composite proteins, plasma, serum, blood, urine, tears, saliva). This article highlights how morphological patterns are influenced by the nature of the biological entities and the solvent, micro- and global environmental conditions. Correlations between emergent patterns and the initial droplet compositions enable the detection of potential clinical abnormalities when compared with the patterns of drying droplets of healthy control samples, offering a diagnostic blueprint. Recent experimental investigations of pattern formation in the bio-mimetic and salivary drying droplets, relevant to COVID-19 are also presented. Finally, we summarize the role of biologically active agents in drying process, including bacteria and algae during the drying process. The review concludes with a perspective on the next generation of research and applications based on drying droplets, enabling potential innovations and tools to study this exciting interface of physics, biology, data sciences, and machine learning., Comment: 92 pages, 22 figures

Biological systems, by nature, are highly complex. These systems exhibit diverse hierarchical spatial and temporal features when driven far from equilibrium. The generated features are susceptible to the initial conditions that largely depend on vast parameter space. Extracting information on their properties and behavior thus becomes far too complex. This work seeks to examine the drying kinetics of the drops containing a globular protein (lysozyme (Lys)), phosphate buffer saline (PBS), and thermotropic liquid crystal (LCs). The drying evolution and the morphological crack patterns of these drops are examined using high-resolution microscopy, textural image analysis, and statistical methods. This study observes that the textural parameters can identify the (i) phase separation of the salts present in the PBS and (ii) the LCs’ birefringence during the drying evolution. This birefringence activities of the LCs slow down when the initial PBS concentration is increased from 0.25 to 1× despite using a fixed volume of LCs. To comprehend such a surprising effect, the combinations of (i) Lys+PBS and (ii) PBS+LCs are thoroughly examined. A phase diagram is established as a function of initial concentrations of Lys and PBS. The scanning electron microscopic images of Lys+PBS reveal that the tuning between lysozyme and salt concentrations in PBS plays a significant role in determining the morphological patterns. The Lys drops with and without LCs exhibit two distinct regions: the peripheral ring (“coffee-ring”) and the central ones. This phase-separated ring formation indicates that the film containing Lys and salts might have formed on top of these LCs in the central region, which reduces the optical response (birefringence) of LCs. A physical mechanism is proposed in this paper to anticipate the redistributions of LCs in a multi-component system such as Lys+PBS+LCs.

Pattern formation in drying protein droplets continues to attract considerable research attention because it can be linked to specific protein–protein interactions. An extensive study of the drying evolution and the final crack patterns is presented, highlighting the concentration dependence (from 1 to 13 wt%) of two globular proteins, lysozyme (Lys) and bovine serum albumin (BSA), in de-ionized water. The drying evolution starts with a constant contact radius mode and shifts to a mixed mode where both fluid front and contact angle changes. The contact angle monotonically decreases, whereas, the fluid front exhibits two regimes: an initial linear regime and a later non-linear regime. Unlike the linear regime, the non-linear regime is faster for Lys droplets. This results in the formation of a “mound”-like structure in the central region. A new feature, a “dimple” is observed in this mound which is found to be dependent on the initial concentration. The different crack morphology of BSA and Lys depends strongly on the initial state of the solution and can be interpreted using a simple mechanical model. In fact, when dried under uniform conditions (surface, humidity, temperature, droplet diameter, etc.), the evolution and the final pattern displays as a fingerprint of the initial state.

The drying process, self-assembly of the proteins and the phase separation of a thermotropic liquid crystal (LC) from an initial aqueous solution represent a rich area of study. A focus of this work is to compare the behavior of two different proteins, bovine serum albumin [BSA] and lysozyme [Lys] in the ternary system through optical microscopy. During the drying process, the intensity profile shows three regimes in the presence of LC whereas no intensity variation is observed in its absence in both protein drops. The striking outcome is the presence of an umbilical defect of [+1] strength in every domain near the edge of BSA drop, whereas, each domain has a central dark region surrounded by a bright region in the dried Lys drop. Finally, the crack spacing in the dried Lys drop is reduced in the presence of LC whereas, no significant difference is found in the dried BSA drop.

This paper discusses the unique patterns evolved through phase separation of a bulk liquid crystal (LC) from the self-assembly of lysozyme induced by evaporation of de-ionized water only. Each domain shows a central dark region surrounded by bright regions (randomly oriented LC droplets). The birefringence intensity reveals three regimes (a slow increase, rapid rise, then saturation) not seen without LC droplets. The textural study exhibits a simple exponential behavior that changes as a function of LC concentration. Furthermore, in the presence of LC, the crack patterns are found to be different near the drop edge than those in the central region.

A bio-colloidal drying droplet can be used as a pre-diagnostic technique. However, a successful clinical setting requires a fundamental understanding of the final morphology and the way it is related to the initial state of the constituents present in the droplet. This chapter focuses on the physics associated with different pattern formations in the globular protein, bovine serum albumin (BSA) at different phosphate-buffered saline concentrations. The study reports that the first-order statistics (FOS) and the gray level co-occurrence matrix (GLCM) analysis are capable of capturing structural changes of the droplets. While the FOS of the image depends on the individual pixels, the GLCM summarizes both tonal and structural relationships between the neighboring pixels. The horizontal and the vertical orientations of the GLCM parameters show a non-significant effect when the pixel displacement is $\leq$ 1. Interestingly, two local equilibrium-like regions (the rim and the central regions) appear when these droplets approach the steady-state. The bimodal distribution confirms that the BSA-BSA interactions are dominant (recessive) over the BSA-saline interactions in the rim (central) regions that result in the phase-separated aggregation of the BSA particles in the presence of the salts.

Multi-colloidal systems exhibit a variety of structural and functional complexity owing to their ability to interact amongst different components into self-assembled structures. This paper presents experimental confirmations that reveal an interesting sharp phase transition during the drying state and in the dried film as a function of diluting concentrations ranging from 100% (undiluted whole blood) to 12.5% (diluted concentrations). An additional complementary contact angle measurement exhibits a monotonic decrease with a peak as a function of drying. This peak is related to a change in visco-elasticity that decreases with dilution, and disappears at the dilution concentration for the observed phase transition equivalent to 62% (v/v). This unique behavior is clearly commensurate with the optical image statistics and morphological analysis; and it is driven by the decrease in the interactions between various components within this bio-colloid. The implications of these phenomenal systems may address many open-ended questions of complex hierarchical structures.

Image processing and pattern recognition offer a useful and versatile method for optically characterizing drops of a colloidal solution during the drying process and in its final state. This paper exploits image processing techniques applied to cross-polarizing microscopy to probe birefringence and the bright-field microscopy to examine the morphological patterns. The bio-colloidal solution of interest is a mixture of water, liquid crystal (LC) and three different proteins [lysozyme (Lys), myoglobin (Myo), and bovine serum albumin (BSA)], all at a fixed relative concentration. During the drying process, the LC phase separates and becomes optically active detectable through its birefringence. Further, as the protein concentrates, it forms cracks under strain due to the evaporation of water. The mean intensity profile of the drying process is examined using an automated image processing technique that reveals three unique regimes: a steady upsurge, a speedy rise, and an eventual saturation. The high values of standard deviation show the complexity, the roughness, and inhomogeneity of the image surface. A semi-automated image processing technique is proposed to quantify the distance between the consecutive cracks by converting those into high contrast images. The outcome of the image analysis correlates with the initial state of the mixture, the nature of the proteins, and the mechanical response of the final patterns. The paper reveals new insights on the self-assembly of the macromolecules during the drying mechanism of any aqueous solution.

The drying of bio-colloidal droplets can be used in many medical and forensic applications. The whole human blood is the most complex bio-colloid system, whereas bovine serum albumin (BSA) is the simplest. This paper focuses on the drying characteristics and the final morphology of these two bio-colloids. The experiments were conducted by varying their initial concentrations, and the solutions were dried under various controlled substrate temperatures using optical and scanning electron microscopy. The droplet parameters (the contact angle, the fluid front, and the first-order image statistics) reveal the drying process’s unique features. Interestingly, both BSA and blood drying droplets’ contact angle measurements show evidence of a concentration-driven transition as the behavior changes from non-monotonic to monotonic decrease. This result indicates that this transition behavior is not limited to multi-component bio-colloid (blood) only, but may be a phenomenon of a bio-colloidal solution containing a large number of interacting components. The high dilution of blood behaves like the BSA solution. The ring-like deposition, the crack morphology, and the microstructures suggest that the components have enough time to segregate and deposit onto the substrate under ambient conditions. However, there is insufficient time for evaporative-driven segregation to occur at elevated temperatures, as expected.