Optical transducers have great potential in the real-time detection of small molecules, but also face intrinsic challenges to fully utilizing their advantages. Optical transductions can measure a variety of signals related to the presence of small molecules, such as absorption, scattering, luminescence, or refractive index
[2]. Optical biosensors typically offer fast results with high temporal resolution for real-time monitoring of binding, making them ideal for diagnostics and point-of-care devices
[7]. However, the signal of optical transducers generally scales with the size and/or mass of the analyte of interest, especially when avoiding the use of fluorescent labels
[2][7][21][2,7,24]. For example, one of the commonly used label-free optical detection techniques, surface plasmon resonance, has been lauded as the gold standard for molecular binding kinetic measurement, being used for analytes such as proteins, DNA, RNA, peptides, and other biological macromolecules, but attempts to utilize SPR for the detection of small molecules required advanced instrumentation or significant enhancement of receptor surface
[15][22][15,25]. Thus, the development of optical techniques for the measurement of small molecules has primarily focused on either enhancing the weak signal of small-molecule binding or by measuring binding indirectly via measuring phenomena induced by analyte-binding events.
Surface plasmon resonance utilizes surface plasmons, electron oscillations at a metal-dielectric interface, typically gold-coated glass, which respond via oscillation at resonance with a light wave
[23][26]. The evanescent waves of this oscillation are sensitive to changes close to the metal surface, notably as a change in refractive index due to the binding of an analyte, which shifts the SPR signal and produces a signal proportional to the analyte’s mass
[24][27]. Unfortunately, this signal dependency on the mass of an analyte makes it challenging for SPR to detect small molecules, often requiring enhancement of either binding site density or signal strength such as through the usage of dextran chips or by utilizing localized surface plasmon resonance with nanostructures
[15]. Thus, there has been great interest in developing sensing platforms with high sensitivity to small molecules, requiring simpler sample preparation and instrument operation, and for diagnostic use, having high portability and low cost.
3. Electrochemical Transduction
Electrochemical transducers share many of the advantages of optical transducers in that they also offer fast, real-time measurements of binding and have the added benefit of generally requiring low-cost instrumentations that can be designed for high portability and accessibility for use with little to no training. Furthermore, the signal of electrochemical transducers is generally not significantly dependent on the size or mass of analytes, but rather on their electronic properties, such as electrochemical reactivity or charge
[16][28][29][16,49,50]. Traditionally, electrochemical biosensors utilized potentiometry or amperometry to detect redox reactions at an electrode surface, offering a mass-independent signal, but this greatly limits the range of molecules that can be detected via electrochemical biosensors to electroactive molecules only. Thus, although real-time electrochemical detection of oxygen and glucose has already reached the commercialization stage for diagnostics and point-of-care monitoring, the detection of other small molecules through electrochemical detection is still developing
[30][47]. For this reason, innovations in the field have explored other means for analytes to modify the electrical properties of transducers. In particular, impedance-based transducers are promising alternatives that are primarily dependent on the charge of small molecules. Thus, impedance biosensors such as FET sensors have grown in popularity for the detection of small molecules due to their detection mechanism, in which the signal is produced by a change in conductance upon binding of charged molecules, allowing high-sensitivity mass independent measurement
[17][18][17,18]. Unfortunately, charge-sensitive techniques face challenges from nonspecific binding and Debye screening from the biologically relevant high ionic strength that hinder their sensitivity in serum, plasma, or even phosphate-buffered saline (PBS)
[6][31][6,51]. Sensing platforms for small molecules have developed methods to use electrochemical transduction that can detect a larger range of different analytes by enhancing the signal strength of impedance-based biosensors to compensate for charge screening in biologically relevant ionic concentrations. Furthermore, enhancement of signal strength increases the sensitivity of electrochemical instrumentation, and helps to detect the relatively low abundance small molecules typically have in nature
[32][33][52,53].
One of the weaknesses of electrochemical detection, particularly in amperometry or potentiometry, is that the analyte of interest must be electrochemically active in order to produce a signal. One workaround to this weakness adapted commercially personal glucose meter for the detection of ATP by using a cascade enzymatic reaction promoted by hexokinase and pyruvate kinase
[34][54]. The amount of ATP is inversely proportional to glucose through the catalyzation of glucose to glucose 6-phosphate in which ATP is converted to ADP
[34][54]. Pyruvate kinase catalyzes the regeneration of ATP from ADP to further react glucose and amplify the signal. Concentrations of ATP as low as 2.5 × 10
−8 g/mL can be detected
[34][54]. This technique offers a potential means to indirectly measure small molecules that are not electroactive by instead measuring the proportional signal of an electroactive product from an enzymatic reaction. Kurbanoglu et al. developed a methimazole (MT) enzyme cascade blocking biosensor using a nanocomposite of magnetic nanoparticles and iridium oxide nanoparticles on screen-printed electrodes and obtained an LOD of 6.85 × 10
−10 g/mL
[35][55]. By utilizing the inhibition of tyrosinase via chelating copper and forming thioquinone with MT, the concentration of MT can be measured via amperometry resulting in a miniaturized lab on a chip biosensor that can be adapted to other small molecules that can inhibit enzymes
[35][55].
4. Piezoelectric Transduction
In comparison to optical and electrochemical transducers, piezoelectric transducers are a relatively recent addition to the repertoire of techniques for detecting small molecules, most popularly utilizing quartz crystal microbalances (QCM)
[19]. Piezoelectricity, the phenomenon in which a material produces voltage under mechanical stress or vice versa, allows for the fabrication of sensors that utilize anisotropic crystals that oscillate upon the application of voltage
[19][36][19,70]. Piezoelectric biosensors typically measure change in oscillation due to analyte binding for the measurement of analyte properties and kinetic information
[36][70]. For example, in a conventional quartz crystal microbalance, the added mass upon binding increases the damping of the oscillation and a change in dissipation rate upon ceasing of voltage application that is related to the mass of the bound analyte following the Sauerbrey Equation
[20][37][20,71]. Unfortunately, Piezoelectric transducers find difficulty in the measurement of small-molecule binding due to the mass dependency of the oscillation’s frequency change; additionally, though piezoelectric transducers are resistant to interference from non-transparent mediums compared to optical transducers, they are responsive to changes in viscosity
[20]. Nevertheless, piezoelectric biosensors can be versatile and robust methods for small molecule detection and much progress has been made in enhancing the sensitivity of piezoelectric-based techniques. Furthermore, most relevant biosensing conditions require the sensor to be in liquid, which produces an additive damping to the measured frequency. Thus, the development of piezoelectric biosensors for small-molecule detection has been in amplifying the change of frequency upon binding or utilizing alternative means to collect data from the piezoelectric transducer.