1. Introduction

Biosensors are an extremely ample R&D area, with applications in many diverse sectors. At present, biological and biochemical laboratories possess a panoply of instruments and detection procedures that provide qualitative detection and quantitative measurement of almost any target, from ions and molecules to full microorganisms like bacteria and viruses.

A quite different scenario, however, shows up when immediate results are needed. In this case, the detection must be done in the field or at the Point of Care (POC) where the target is generated or scrutinized. Often the detection is carried by non-specialists. POC equipment must be portable, and tests should ideally be simple, sensitive, fast and carried out with low power consumption –ideally powerless– using inexpensive disposable elements. These restrictions often hinder the use of sophisticated equipment and lead to new proposals of alternative procedures for POC controls.

The European project Raptadiag [1] had the overall objective of finding simple, reliable biosensors and procedures for POC detection of pathogens (specifically Meningococcus, but the results could be extended to other microorganisms). The approach was to test three different detection procedures –gravimetric sensors made of piezoelectric crystals or thin-films, laser-induced evanescent fluorescence, and liquid crystals–, while aptamers and antibodies were employed for selective binding of targets through surface functionalization. Aptamers are short single-stranded oligonucleotides from DNA or RNA have unique 3D structures which are highly selective and have high affinity towards a desired target [2,3]. Compared to conventional antibodies, aptamers are more thermally stable, less costly, and faster to develop and to produce.

This work focuses on detection with liquid crystals (LCs). Liquid crystals are a state of the matter with intermediate properties between solids and liquids: they are ordered and birefringent –properties typically associated to solids– but they can flow, like liquids. LCs are anisotropic for a number of physical properties including viscosity, dielectric permittivity, magnetic permeability and optical refractive index [4]. This has paved the way to applying LC materials to a startling number of applications, among them electronic and photonic devices for modulation and control of light and other electromagnetic waves like THz and microwaves [5]. Additionally, LCs show an extraordinary sensitivity to external electric and magnetic fields, as well as changes in boundary surface conditions which may be transduced into optical signals, visible between in polarized light.

Liquid crystal biosensors usually take advantage of this sensitivity. A typical LC biosensor detects the presence of small molecular compounds by adsorbing them on an aqueous/lipidic interface. LCs can also detect microparticles of a certain size. In either case, the visual signal usually arises from disordered LC regions of an otherwise ordered LC, due to the presence of the target. The ordered LC can be made dark between crossed polarizers, while disordered regions show up as bright colored spots.

An interesting feature of LC sensors is amplification [6]. The disorder propagates to an area considerably larger than the original target size, making it possible to observe submicron- or micron-sized targets with a standard polarized microscope; in some cases, amplification can be high enough to allow defects to be visible at naked eye. Different LC materials and procedures have been proposed for biosensing of DNA [7], enzymes [8], antigens [9] and even full microorganisms like viruses and bacteria [10], as well as biologically relevant compounds such as environmental pollutants and gases [11].

Most proposed LC biosensors are based on nematic LCs [12] and other thermotropic liquid crystals, like cholesterics [13,14], blue-phase [15], and smectics [16]. All these LCs feature a common issue, their immiscibility in aqueous media. As many biological samples are solved or suspended in water, biosensing with thermotropic LCs requires alternative solutions to achieve interactions between targets and LCs [17]. Free-standing LC droplets [18,19] or surfaces suspended in sub-mm grids [20] have been proposed to procure an interface for interactions between the hydrophobic LC layer and the aqueous sample being examined [21]. The LC spontaneously orients homeotropically (perpendicular) to the free-standing surface for surface tension. Adsorption of targets on the interface modifies such orientation, and the change can be observed in a microscope between crossed polarizers.

A different straightforward approach to overcome these difficulties is to use lyotropic, rather than thermotropic, liquid crystals. Lyotropic liquid crystals (LLC) are stable and soluble in aqueous media since they mostly derive from amphiphilic molecules. LLC phases exist in a range of temperatures, and within a range of concentrations of the material in a solvent, usually water. LLCs are ubiquitous in biological media, forming for example the lipidic bilayer of cell membranes. However, their interest as materials for technical applications is largely overtaken by thermotropic liquid crystals, particularly calamitic nematics. Nevertheless, water based lyotropics are the preferred alternative in some biological applications, in general, those whose actual working conditions imply the use of hydrophilic media [22]. LLCs have been proposed, for example, as biomimetic vehicles for delivery of sparingly soluble drugs or medical contrast agents [23].

The use of LLCs for biosensing of microparticles and microorganisms is known for some time [24]. In this case, the aqueous solvent where the targets are suspended is mixed with the LLC –taking into account the concentration range where the desired lyotropic phase is formed. A cell is formed by two transparent surfaces (glass, rigid polymers) with a small gap (some µm) between them. The inner cell surfaces are conditioned in advance to induce a certain orientation on the LLC. The element can be integrated in a microfluidic system. In the absence of targets, surface preconditioning makes the LLC to orient as the surfaces dictate. If targets are present, they may induce defects in the overall orientation; these defects can be visualized between crossed polarizers in a microscope.

Visualizing director defects is possible when the object in the medium is larger than the extrapolation length [17]

To achieve specific sensing it is necessary, after adding the alignment layer, to functionalize at least one of the inner surfaces of the cell with some reagent specific for a given target, e.g., an antibody, and to include a washing process in the sampling protocol. Bound microorganisms are then revealed as defects in alignment. An alternative procedure is to coat only one of the plates with the alignment layer and to employ the other plate as functionalized surface. This requires the alignment anchoring to be strong enough for the sample to be aligned with just one surface.

In this work, the optimization of LLC biosensors for selective detection of microorganisms has been undertaken. Several aspects have been considered: seeking alternatives of antibodies for selective detection, providing good alignment conditioning for LLCs, and increasing the amplification, with the ultimate goal of performing the test without optical aids, i.e. with naked eye.

2. Experimental

The optimization of LLC biosensors includes the selection of binding agents, manufacturing of alignment layers, and enhancing amplification. Anyhow, the aim of this work is to evaluate the performance of LLCs in biodetection; this will wrap up the whole set of activities.

2.1 LLC cell manufacturing

LLC cell geometry is the same as in standard LC cells with a few differences. Two parallel glass plates separated 10 µm were assembled using a photocurable adhesive. Opposite ends of the cell were left open to allow filling by capillarity or microfluidics. Spacers were only added to the adhesive layer framing the cell, since any microparticle inside the cell would lead to a false positive.

One of the inner surfaces was functionalized either with an aptamer or an antibody so that a specific pathogen would be bound to the surface. The other surface was used to induce an alignment to the LLC. The alignment was homogeneous –i.e., parallel to the surfaces– and oriented along the flow direction when the cell is filling up.

2.2 Lyotropic liquid crystals

Two LLCs have been chosen for the experiments: Sunset Yellow FCF (disodium 6-hydroxy-5-[(4-sulphonatophenyl)azo]naphthalene-2-sulphonate) and Cromolyn (sodium cromoglycate) (Sigma-Aldrich). These two materials are among the most studied LLCs. Both are water-soluble salts of organic acids, and possess a nematic (N) phase at room temperature in the concentration range 27% < N < 35% w:w for Sunset Yellow (SSY) and 13% < N < 17% w:w for Cromolyn. Despite Cromolyn is the most used and studied, the work ultimately focused on SSY since it showed several advantages: a wider nematic range, a lower variation of this range with temperature, and a lower viscosity, which resulted essential for dynamic studies shown below.

2.3 Aptamers

Most of the work on LLCs has been carried out employing aptamers [26]. Specific aptamers (80–120 bases) for Meningococcus (the original target), and foodborne pathogens such as Salmonella, Listeria, and Campylobacter have been prepared within the project by the iterative SELEX method [27]. Legionella, prepared outside the project, has been employed as well. Once sequenced and replicated, aptamers were bound to one inner surface of the LLC cell, making the surface functional to specifically trap a target –in our case, a pathogen. Details of fabrication and characterization of these aptamers are beyond the scope of this work.

2.4 Alignment layers

Finding a surface conditioning coating for inducing orientation of an LLC is not a trivial task. Standard polyimides employed in thermotropic nematics do not work adequately with LLCs, nor polyamides (e.g., Nylon 6) used in nematics and smectics. A study for finding the most suitable alignment layers for selected lyotropics was carried out. The results have been published elsewhere [28,29].

2.5 Amplification

Increasing the amplification of the detected targets –i.e., the area where the LLC orientation is affected, hence giving positive signals– is utmost important for the realization of practical devices. Indeed, larger areas imply that the signals are more easily detected. There is a threshold, however, above which the defects are seen with naked eye. Achieving this threshold produces a significant change in the detection procedure: the experimental setup reduces to a mere low-cost disposable cell, making it much easier the spreading of the method, and allowing the use of multiple tests in different places, since the time for assay reduces as well.

Consequently, the main scope of this work has been to achieve an amplification factor significantly higher. The development of a new dynamic procedure of detection has accomplished this goal, as shown in the Results & Discussion section.

3. Results and discussion

3.1 Static preliminary measurements

We have tested the quality of LLC orientation with inorganic and organic layers. Tested inorganic layers were silicon oxides SiO₂ and SiO_x deposited by tilted e-gun and thermal evaporation respectively. Both oxides showed an ample angular range where good alignment was achieved. The range was similar for both LLCs and both oxides; the SSY/SiO₂ was the best option. The orientation was homogeneous in all cases. The tilt angle for evaporation of all samples was set in the range 50°–60° (Fig. 1).

 

Fig. 1 An SSY/SiO₂ homogeneous cell between crossed polarizers. Left: LLC indicatrix at 45° of polarizer axes; right: LLC indicatrix parallel to polarizer axes. The evaporation angle was 55° in both substrates.

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After developing several specific aptamers mentioned above, we have been able to detect pathogens using these aptamers as functionalizing agents onto one of the inner cell surfaces. Detection has been performed on two kinds of fluids: those related to food chains (milk, water), and body fluids like saliva, lymph, urine, plasma, or cerebrospinal fluid. The tests are not intended to substitute standard analyses of healthcare labs, but rather be used as screenings for POC tests; any possible positive detection would be eventually confirmed through a full analysis.

Nevertheless, our rates of false positives with aptamers are still unacceptable for actual working biosensors; this issue is under study. The results of this work have been obtained employing either antibodies or artificial samples with just one, or a few, kinds of microorganisms. Figure 2 shows preliminary results testing the detection procedure. A 10µm thick SSY/SiO₂ cell was filled with a clean solution and with a doped solution. No surface functionalization has been performed.

 

Fig. 2 Microscopic view of SSY/SiO₂ sample without (left) and with targets.

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The targets were silica microspheres of 2.5 µm diameter. Targets are clearly visible as bright spots in a dark field (the residual brightness of the undoped sample is due to the automatic gain of the camera; actually both pictures are equally dark). The visible field width is about 1 mm. The estimated size of the spots is 20–25 µm; therefore, the amplification factor is about 8–10. Similar results have been published by other authors [10].

The targets, or the agglomerates in the case of functionalized surfaces, produce a disorder in the neighboring LC molecules since they must adapt to the new anchoring condition imposed by the target [24]. Disorder propagates a certain distance while the influence of the anchoring energy fades out. Indeed, the distance would depend on several external parameters, such as LLC concentration and temperature, the solution’s ionic strength and, obviously, the physical characteristics of the targets and their size. However, for a given target and a specific set of experimental conditions, the distorted area, hence the amplification factor, is apparently fixed.

3.2 Measurements under flow: wake formation

In the above scenario the LLC organization and orientation are static, i.e., performed once the cell has been filled up with the mixture of target and LLC solutions. The situation is drastically modified if the LLC is studied under a controlled flow. If the sample is homogeneous, and the imposed orientation is parallel to the flowing direction, the LLC spontaneously orients upon flowing. This phenomenon [30] was known since the dawn of liquid crystals and is due to the anisotropy of LC viscosity.

When this (partially) oriented flow finds obstacles (i.e., targets) to the flow progression, a distortion is produced. Contrary to the static case, the disorder propagates asymmetrically, the distorted length in the direction of flow being orders of magnitude longer than the normal component. The effect is similar to the wakes generated by a ship in calmed waters, or the trails of jet planes. Eventually the wakes fade away (Fig. 3); the time elapsed between the wake formation and fading is linked to the flowing speed of the solution; it is usually in the range of minutes.

 

Fig. 3 Dynamic detection. The same cell and targets as in previous figure have been used. The flow runs for right to left. The pictures were taken at 30s, 60s, and 90s respectively from left to right. The visible field is about 2 mm. Note that the field is not dark since the LLC is only partially oriented.

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As seen in the figure, wakes of about 1 mm are formed. The most important consequence is that dynamic detection is achieved by naked eye, considerably simplifying the required experimental setup for field tests [31]. The flow enhanced amplification factor is about 400–600, i.e., almost two orders of magnitude higher than the factor in static measurements.

Figure 4 is a display of the whole process with time. The LLC solution was introduced by capillarity. Pictures in the top row were taken during filling, while pictures in the bottom row were taken after the cell was completely filled up, stopping the flow. The wakes develop as soon as the filling starts (top left); however, they are barely visible since the LLC is still quite disordered, hence crossed polarizers do not prevent light propagation through the cell. After a few minutes (top right) the flow becomes steadier as the LLC molecules become more oriented, darkening the cell and allowing the wakes to show up. Note that flow is still active.

 

Fig. 4 Formation and fading of wakes within and after the cell filling.

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Once the cell is filled up, the flow stops, and the wakes begin to vanish. Yet they still persist a short time (bottom left). A few minutes later (bottom right) the wakes have disappeared, just leaving bright dots similar to those of the static case.

3.3 Microfluidic system

As mentioned above, wake formation and persistence are closely related to the LLC flowing rate, which in turn depends on the solution viscosity and the geometrical dimensions of the cell. In actual working conditions, these parameters would be adequately adjusted; however, when testing new mixtures and/or geometries, it should be convenient to have control on flow for a variety of conditions. This can be achieved including the cell into a microfluidic system attached to a peristaltic pump. Figure 5 shows the simple microfluidic system employed in the experiments of this work. It consists of two symmetric polymer pieces with prismatic cavities and two connecting cylindrical tubes for fluid input and output from the external circuit. Note that the two pieces for fluid input and output are separated, leaving a central region for optical inspection.

 

Fig. 5 Left: simple microfluidic system for insertion of cells with control on injection pressure. Right: cell in working conditions, with input/output needles connected.

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Test cells for actual working conditions were prepared and inserted in the microfluidic system [32]. Several aptamers were tested (Legionella, Salmonella, Meningococcus) attached to the functional surface of the cell through an APTES/Aptamer procedure described elsewhere [33]. As commented above, detection of microorganisms has been obtained in all cases, but our rate of false positives is still too high when working with actual fluids. By now, cells work correctly when only one single type of microorganism, or just a few, are introduced and bound to antibodies (AB). Figure 6 shows an example in which cells have been functionalized with a Legionella antibody (AB) (positive) or with an irrelevant AB, in this case, Mouse AB (negative). The cells are, then, loaded with a dilute solution of Legionella. After incubation for 30 minutes, where Legionella is selectively fixed to the corresponding ABs, SSY was filled into the cells at a constant flowing rate to wash off the solution. After 10 minutes, the solution containing Legionella has been completely substituted by SSY and thus LC’s birefringence shows up. At that point, long wakes start being visible between crossed polarizers.

 

Fig. 6 Dynamic Legionella detection in a liquid crystal flow: surfaces functionalized with Leg AB bound many more cells than the ones functionalized with Mouse AB. The central area close-ups (right) show a clear difference between both samples. A few false positive wakes, attributed to microparticle impurities, are seen in Mouse AB.

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The cell on the left side shows the long wakes, as Legionella pneumophila is detected where it has been bound to the surface, while the cell on the right shows little or no wakes given that Mouse ABs do not bind Legionella. The few wakes observed in the negative cells are attributed to surface defects or microparticles and non-specific interactions during functionalization of the surface. Anyhow, there is a relevant difference between both samples to the naked eye. The size of the active area is 18 × 8 mm; the close-ups on the right side of the figure show the central area of the cells (6 × 5mm).

4. Conclusions

A new procedure for detection of macromolecules and microorganisms has been described. The procedure is simple, inexpensive and semiquantitative. Different targets can be explored, even in the same cassette. The procedure may be useful for point-of-care fast tests carried out by non-specialists. The remarkably high amplification factor achieved in dynamic monitoring allows in most cases to detect the target by naked eye, further simplifying the required experimental setup.

Lyotropic liquid crystals reveal as a strong alternative for liquid crystal-mediated biosensors, especially in those cases were a hydrophilic medium must be used, like milk or animal waste in food chains or sea water in aquaculture. They are also a good candidate for biosensors utilized in body fluids like saliva, lymph, urine, plasma, etc.

At present, the tested procedures have shown a significant number of false positives, while false negatives are nearly absent in correctly functionalized cells. It may be argued that false positives are less relevant than false negatives. Indeed, the test can be used in many applications as a simple screening to select those samples or patients that should be analyzed in an eventual control at a healthcare center. In these cases, a certain number of false positives, if not excessive, can be acceptable, while false negatives must be avoided in any circumstances.