Types of biosensors in contact lenses.
Introduction:
The rapid advancement of
technology has enabled the realization of personalized medicine. According to
the global demographics of health reported by the World Health Organization,
75% of the world population is defined as sub-health, but only 20% needed to be
hospitalized. As the sub-health population has become more concerned about
their quality of life, the demand for so-called point-of-care (POC) diagnostics
is rising. tear fluid contains relatively rich proteins that have been
identified and share similar constituents to that of blood. This similarity is
attributed to the plasma leakage across the blood-tear barrier and thus enables
the development of biosensors that specifically target analytes within the tear
fluid. Although blood testing remains second to none in the standard liquid
biopsy, tear fluid can be collected in a non-invasive way. This feature makes
tear fluid a promising sample in some preliminary clinical tests. Followed by
this concept, contact lenses have become an ideal platform for such kind of
diagnosis as considering their irreplaceable popularity in our life. An
emerging trend shows that
researchers have begun to put more emphasis on the diagnostic potential of contact lenses with various lacrimal biomarkers. Up to now, contact lenses have been successfully integrated with sensors for continuous monitoring of diabetes and glaucoma. The attempted development of contact lens biosensors gradually sparks commercial interests and initiates a brand-new market.
A recently eye-catching collaboration between Google and Novartis came up with a smart contact lens for diabetes management. Ultimately, the integration of wearable devices with the Internet of Things and patient records will allow for more efficient implementation of treatment strategies Despite the promising future, contact lens biosensors remain in their infancy. Emerging challenges may come from long-term durability, the robustness of driving mechanisms, ease of use, and even clinical perspectives. These factors may determine this new market to rise or fall in the end. In this review, we particularly focus on tear-based techniques with biomarkers for disease diagnoses. At last, their latest applications in contact lens biosensors will be explicitly addressed as well. body fluids, tear (or lacrimal) fluid is a complex multilayered concoction of proteins, lipids, enzymes, and salts. As a result, a variety of biomarkers are present in the fluid and can be potentially used for disease screening. In general, the tear film consists of three major layers that function as a lubricant and cleansing agent for the eye. Notably, the tear fluid, in part, shares compositional similarities with the blood, because of plasma leakage across the blood-tear barrier. Although the majority of components differ between the two body fluids, the plasma leakage allows examinations of the tear fluid instead of blood. Prior studies have shown that components, such as glucose, Na+, K+, and Cl− ions, can be found in both the blood and tear fluid. Nonetheless, tear fluid is proven to be a less complex body fluid as compared with serum or plasma because of the blood-tear barrier. This strong filtering effect makes tear fluid contain significantly fewer proteins than its blood counterpart. The reported number of proteins detected within the tear fluid is believed to be between 54 and 1543. The variation in the number of proteins can
be highly dependent on the sampling method. In this regard, there are three important tear types that can alter the composition of tears: (1) basal—the protective tear fluid produced during the normal daily operation of the eye that covers the corneal surface; (2) reflex—secreted due to physical or chemical stimulation and to help remove irritants such as foreign particles, vapors, and gas, bright lights, or the action of coughing or yawning; and (3) psychic—induced by emotions (anger, pleasure, or pain), and contains higher concentrations of hormones. Sampling methods can affect the composition of the tear fluid collected. For example, Schirmer strips are likely to be in contact with the cornea; resulting in reflex tear secretion, alternatively, microcapillary tubes can be used. Although the use of these tubes is less invasive than Schirmer strips, however, the collection process remains time-consuming. Both of these methods are also limited to low volume tear collection, such that they become problematic when it comes to patients with dry eye disease.
Type of biosensors
- Contact lens biosensors
The identification of
potential biomarkers for the early detection of diseases and disorders is still
ongoing. Currently, known biomarkers are being tested in biosensors
for POC applications, which range from on-chip sensors to functionally integrated sensors, such as contact lenses. Since a large market has taken shape in diabetes management, research and development of contact lens sensors are predominantly focused on the glucose-related field. As the sensing technology advances, the range of diseases that can be monitored and detected via contact lens biosensors will increase. Continuous monitoring may not be required for many diseases, and contact lens biosensors can also serve as a one-time use diagnostic tool. In this case, contact lenses will naturally accumulate tear components during wear and can be analyzed after wear. By integrating the detection of specific biomarkers, such as in cancer, or dry eye, it would be possible to identify the presence and progression of certain diseases. This section lays emphasis on the major emerging methods of detection, such as fluorescent, holographic, colorimetric, and electrochemical, based on contact lenses. Performance comparison of different types of contact lens biosensors is detailed in Cutting-edge developments that may have the potential for integration will be discussed in the following as well.
- Fluorescence – based sensing
Fluorescence-based sensing has been utilized in a plethora of applications owing to its versatility, sensitivity, and specificity. The basis of fluorescence is the absorption of electromagnetic radiation of a specific wavelength by an excitable fluorophore and the subsequent emission of photons with longer wavelength. The released photons can then be differentiated from the background noise with filtering techniques, making this detection technique highly sensitive. The excited and emitted photonic wavelengths are dependent on the chemical structure of the fluorophore, which allows customizable and highly specific fluorescence sensing. About contact lens applications, a biosensor for glucose detection through immersion in tear fluid was developed early on by March et al. with tetramethylrhodamine isothiocyanate concanavalin A (TRITC-Con A) and fluorescein isothiocyanate dextran (FITC-dextran) encapsulated within hydrogel spheres that were embedded and immobilized in polymerized Nelfilcon A (PVA-based) within a contact lens mold.
As glucose diffuses into the spheres, the FITC-dextran molecules are shifted away from TRITC-Con A, which results in a decreased Forster Resonance Energy Transfer (FRET) and the increase of fluorescence intensity. This biosensor was able to track the concentration of blood glucose in patients over three hours. However, a delay of 15 min between the blood glucose concentration and readout from the sensor was found. The biosensor was compatible with a hand-held photo fluorometer, which obtained the green fluorescent readout signal. It was proposed that the photo fluorometer could also be used in conjunction with an insulin pump (or similar) to be used in the management of diabetes. In another study, a contact lens biosensor, using pHEMA or PDMS, embedded with organic dyes encapsulated within silica nanoparticles was proposed. This biosensor was able to detect glucose in the range of 0.5 and 5 mM. The use of silica maintained the capsule shell integrity and prevented premature leakage. Similarly, Badugu and his colleagues developed boronic acid-based probes for tear glucose detection and then successfully integrated them into commercially available contact lenses.
Their study demonstrated that the probes could readily detect tear glucose changes within the range from 50 μM to 100 mM for diabetics. The contact lens biosensor had a response time of 10 min, such that it allowed continuous and non-invasive monitoring of physiological glucose and reducing the need for invasive blood sampling. More recently, the Lakowicz group attempted to fabricate a contact lens platform to evaluate the ion concentrations in tear fluid via fluorescence. The use of commercially available silicone hydrogel contact lenses enabled the binding of hydrophobic ion-sensitive fluorophores. Notably, silicone-based contact lenses can facilitate the rapid transport of both oxygen and tear fluid, hence they are feasible for long-term use. Other than proteins, Badugu et al.demonstrated that functionalizing silicone contact lenses with fluorescent probes could even detect changes in chloride ion concentration and pH. The proof of concept, therefore, paved a way for more probes for use in the detection of sodium, potassium, calcium, and magnesium ion concentrations. The same research group hopes to create a multiplexed platform by functionalizing various regions of a contact lens to measure the concentrations of tear electrolytes. In general, the development of water-soluble fluorescent probes for biosensing offers various advantages such as high specificity, versatility, and potential for easy analysis via handheld readers/detectors. By properly incorporating immunofluorescent assays into contact lenses, it would be possible to detect a wide variety of biomarkers, such that diseases can be diagnosed simply through non-invasive liquid biopsy alone.
- Multiple-Dimensional Photonic Crystal: Colloidal Crystal Arrays
(CCA)
In addition to the one-dimensional PC introduced previously, two- and three-dimensional PCs are tremendously developed for biosensing purposes as well. Photonic crystal array sensors are 3D PCs consisting of nano-sized particles immobilized within polymer matrices. These PCs are formed from orderly-stacked crystalline nanospheres, such as polystyrene or silica, and have been used in biosensors for glucose in tear fluid. The construction of CCA involves the self-assembly of monodispersed particles by taking advantage of the evaporation of the colloidal solutions. Alexeev et al. were one of the first to develop photonic crystals in glucose sensing applications.
Their sensor consisted of polystyrene colloids embedded
within a PA-(bis-AA)-poly (ethylene glycol) matrix and functionalized with
boronic acid derivatives (4-acetamido-3-fluorophenylboronic acid and
3-fluoro-4-N-methyl carboxamide phenylboronic acid), which permitted the
sensing of glucose at physiological pH. The measurement mechanism involves
glucose binding to boronic acid and forming additional linkages within
the polymer matrix, which results in the overall shrinkage and modification of
the lattice spacing. The shrinkage of the matrix is proportional to the glucose
concentration in the tear fluid and results in a blue shift of the diffracted
light. In another study from the same research group, the sensitivity and
response time of the CCA sensors were further improved for the detection of
glucose concentrations in blood (5 mM) and tear fluid (0.15 mM). The optimal
response times for blood and tear fluid measurements could respectively reach
90 s and 300 s at physiological pH and temperature. For the tear fluid, a blue
shift of 11 nm was observed with a detection limit of 1 μM in synthetic tear
fluid.
CCAs formed from microgel spheres in glucose detection was also reported by Wu et al. where glucose-sensitive poly (styrene-co-acrylamide-co-3-acrylamidophenylboronic acid) self-assembling spheres were embedded within a poly (acrylamide-co-2- (dimethylamino) ethyl acrylate) hydrogel matrix. The CCA-embedded hydrogel responded to various glucose concentrations by swelling, with a maximum swell ratio of 2.02 at a concentration of 300 mg/dL, and could fully recover after the dissociation of glucose. The detection of glucose was performed using reflection spectra at 1722 nm (near-infrared), and it was demonstrated that the CCA-hydrogel was highly stable, even after five months of storage at room temperature. Also, interference from non-sugar constituents in tears was found to have minimal impact on the reflection spectra with relative errors less than 3%. The results of the study revealed that the detection limit of The Embedded hydrogel was 6.1 μg/dL, while physiologically relevant glucose the concentration of 7.5 mg/dL could be determined within 22 s and reaching a maximum in 2 min.
Shorter response times were achieved with higher glucose
concentrations. The characteristics mentioned above demonstrated the potential
of this material for a POC device, particularly incorporation within contact
lenses. Although most materials presented here focus on boronic acid
derivatives and glucose sensing, it may be possible to functionalize these
CCA-embedded hydrogels with biomarker-specific antibodies for the detection of
other diseases. Inverse-opal photonic crystals produced from CCAs have
also been investigated for use in biosensing. Choi and colleagues came up with
a Nanoporous photonic crystal structure within a hydrogel for use in the
detection of immunoglobulin G (IgG) antibodies. The fabrication of their device
involved the self-assembly of silica nanoparticles followed by immersion within
a poly (ethylene glycol) diacrylate matrix and UV-curing. The silica
nanoparticles were then etched away to reveal the nanoporous structure within
the polymer matrix. The nanoporous structure was functionalized with Protein A
that bound IgG antibodies and was able to effectively distinguish various
concentrations of IgG antibodies. As the concentration of antibodies increased,
the reflected wavelength red-shifted to a unique peek at each concentration as
a result of a change in the effective refractive index. The results of this
study demonstrated that inverse-opal structures have the potential to be used
in the detection of other analytes, and could be integrated within contact
lenses to form wearable biosensors. However, the sensitivity of this type of
sensor will need to be improved to detect trace amounts
of biomarkers within tear fluid.
- Electrochemical sensing:-
Electrochemical sensors for bio analyte detection have been well-researched over the past decades. Various fabrication techniques, especially those used in the semiconductor industry, are commonly employed to construct biosensors. In the case of glucose detection, the sensors utilize enzymatic action that allows for highly selective reactions to occur for electrochemical sensing. Generally, glucose oxidase (GOD) converts glucose to gluconolactone and hydrogen peroxide, which further dissociates into oxygen, hydrogen ions, and electrons.
The three-electrode system of the sensor utilizes the electrons produced to quantify the concentration of glucose in the fluid. These chip-based electrochemical sensors have also been integrated into contact lenses for the determination of glucose concentration in artificial tear fluid. In this study, the sensor circuitry was fabricated using a photoresist (AZ4620) layer to pattern and deposit Ti/Pd/Pt onto a PET wafer. The resist layer was dissolved in acetone and the wafer was heat-molded into a contact lens, with the electrodes on the convex surface. Pre-
treatment of the sensor required the GOD to be immobilized within a titania sol-gel film and applied to the sensing electrodes before testing of samples. The developed contact lens sensor had a detection limit of 0.01 mM glucose, approximately one-tenth of glucose concentration in human tears Similarly developed a contact lens biosensor for the in situ monitoring of tear glucose using PDMS as the contact lens material and a thin PDMS film as a base for the flexible hydrogen peroxide electrode (with Pt and Ag/AgCl as the working and reference/counter electrodes, respectively) Initial in vitro testing confirmed that the biosensor was able to detect glucose concentration in the ranges of 0.03 to 5.0 mM, which covered the normal tear glucose levels in humans (0.14 mM). Subsequently, the in-situ performance of the fabricated contact lens the biosensor was investigated via introduction to the eyeball of a rabbit model and corroborated with conventional glucose blood tests. The glucose levels within the blood and tears were spiked by the oral intake of glucose solution by rabbit, and it was demonstrated the biosensor was able to detect a change in the tear. glucose levels with a delay of 10 min and peaked after 55 min.
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