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Figure 23.( a) Sensorgram of thrombin binding to a WGMR immobilized with TBA-15 in 1:10 unfiltered human serum( one thrombin addition).( b) Sensorgram of thrombin binding to a regenerated WGMR immobilized with TBA-15( three additions, 5 lL of 0.3 mg / mL thrombin each). Adapted from [ 91 ].
Microspherical WGMR based sensors have been extensively used as biochemical sensors, Giannetti et al. [ 97 ] demonstrated the possibility of using polymeric layers as an alternative to silanes for IgG detection. Zhu et al. [ 98 ] demonstrated the possibility of using aptamers as BRE for thrombin detection. Following this pioneering work, Pasquardini et al. [ 91 ] used microspherical WGMR to detect two different thrombin aptamers and one vascular endothelial growth factor( VEGF) aptamer. Thrombin functions as a coagulation factor and it is involved in many pathological diseases, like atherosclerosis and thromboembolic diseases. Thrombin-15( a 15-mer DNA-aptamer) binds to the fibrinogen exosite and Thrombin-29( a 29- mer one) binds to the heparin exosite of thrombin protein. VEGF is an important regulator of angiogenesis and it promotes the migration and proliferation of endothelial cells and the formation of new blood vessels from preexisting capillaries. The detection of these blood proteins in clinical and laboratory measurements is time consuming and costly, mainly due to the lack of available antibodies. The authors tested the performance of the WGMR aptasensors in a microfluidic flow cell under laminar flow conditions in buffer solutions and in a real matrix, a 10 % unfiltered human serum solution, and they also tested the reusability of the WGMR sensor. The sensor regeneration was done by 50 mM NaOH solution and the WGMR aptasensors were then used for another detection cycle [ 91 ]. Figure 23a shows a typical sensorgram of thrombin binding to a WGMR immobilized with TBA-15 in 1:10 unfiltered human serum, following a single addition of thrombin( red dots). Figure 23b, instead, shows the thrombin binding to a regenerated WGMR immobilized with TBA-15, following three additions of thrombin( 5 lL of 0.3 mg / mL thrombin each).
MBR have also been proposed as biosensors and they are of particular interest since multiplexed sensing can be achieved. Berneschi et al. proposed a procedure for a spatially selective immobilization of antibodies, via photochemical activation, on the inner surface of the microbubbles [ 99, 100 ]. Figure 24 shows the multi-selective immobilization of two different IgG labeled with AlexaFluor647( red) and AlexaFluor488( green).
5.2 Optoplasmonic sensors
Arnold et al. also pioneered the use of hybrid WGMR to enhance the detection of single virions [ 101 ]; a RNA virus
Figure 24. Fluorescence images of two MBRs fabricated on the same capillary.( a) Immobilization of goat anti-rabbit IgG labeled with AlexaFluor647 bound on the inner surface of the MBR on the right side.( b) Goat anti-rabbit IgG labeled with AlexaFluor488 bound on the inner surface of the MBR on the left side.( c) Merging of the previous images. Reproduced with permission from [ 99 ].
Figure 25. The resonance shift curve( black) shows pulses when single Tg proteins adsorb to the gold nanoshell coating the WGMR equator. Blue curve shows the background without the protein nor the gold nanoshell. Insets: optical image of the hybrid WGMR( top left) and resonance profile( bottom right). Adapted from [ 101 ].
MS2 with a mass of 6 ag was detected using the metallic shelled WGMR. One year later, the same authors reported the detection of a single Thyroglobulin( Tg), a thyroid cancer marker, and of bovine serum albumin( BSA) proteins with masses of only 1 ag and 0.11 ag( 66 kDa), respectively [ 102 ]. Figure 25 shows the sensorgram for Thyroglobulin detection, an optical picture of the resonator and the Q-factor of the hybrid WGMR.
Frank Vollmer group has been using the optoplasmonic enhancement to detect biochemical interactions such as conformational changes of DNA polymerase [ 103 ]. The authors studied how a change in enzyme polarisability or volume resulted in a change in WGM resonance frequency. The same group provided evidence for the macromolecular rate theory of enzyme catalysis by measuring negative heat capacity change via maltose-inducible a-glucosidase( MalL) temperature-dependent single-molecule kinetics [ 103 ]. The group has also published a study of the response of