Light-switching excimer probes for rapid protein monitoring in complex biological fluids

Abstract

Quantitative protein bioanalysis in complex biological fluids presents considerable challenges in biological studies and disease diagnosis. The major obstacles are the background signals from both the probe and the biological fluids where the proteins reside. We have molecularly engineered light-switching excimer aptamer probes for rapid and sensitive detection of a biomarker protein, platelet-derived growth factor (PDGF). Labeled with one pyrene at each end, the aptamer switches its fluorescence emission from Ϸ400 nm (pyrene monomer) to 485 nm (pyrene excimer) upon PDGF binding. This fluorescence wavelength change from monomer to excimer emission is a result of aptamer conformation rearrangement induced by target binding. The excimer probe is able to effectively detect picomolar PDGF in homogeneous solutions. Because the excimer has a much longer fluorescence lifetime (Ϸ40 ns) than that of the background (Ϸ5 ns), time-resolved measurements were used to eliminate the biological background. We thus were able to detect PDGF in a cell sample quantitatively without any sample pretreatment. This molecular engineering strategy can be used to develop other aptamer probes for protein monitoring. Combined with lifetime-based measurements and molecular engineering, light-switching excimer aptamer probes hold great potential in protein analysis for biomedical studies.aptamer ͉ biomarker ͉ platelet-derived growth factor ͉ pyrene ͉ time-resolved fluorescence

Specific detection of biomolecules in physiological solutions using graphene transistor biosensors

“…The DNA aptamer is advantageous as the receptor for several reasons, including (i) the conformational changes of highly charged aptamer upon protein binding (37,38) can result in a significant change in electric field near the sensor surface even if the solution pH is close to the protein pI, and (ii) the aptamer can be denatured and refolded multiple times without loss of activity (39,40) for multiple experiments. All experiments were carried out in pH 7.4 1× PBS containing 2 mM Mg 2+ , where the added Mg 2+ helps to maintain the active aptamer conformation before and after regeneration (39,41).…”

Metal‐Induced Specific and Nonspecific Oligonucleotide Folding Studied by FRET and Related Biophysical and Bioanalytical Implications

“…[5] The adaptive binding property has been used to design fluorescence resonance energy transfer (FRET)-based sensors with end-labeled fluorophores since FRET is known to be sensitive to such distance changes. Many molecules include adenosine/ATP, [6] cocaine, [7] arginiamide, [8] K I , [9][10][11][12] Mg II /Ca II , [13] Ag I , [14] Hg II , [15,16] thrombin, [17] and (platelet-derived growth factor) PDGF [18,19] have been detected using this method. One particularly interesting example is the Hg II binding DNA shown in Figure 1A.…”

Flow Cytometry-Assisted Detection of Adenosine in Serum with an Immobilized Aptamer Sensor

“…Among the various signal transduction methods, fluorescence has been the most often used due to its high sensitivity and versatility. 5,[20][21][22][23][24][25][26][27] In addition to intensity-based detection, fluorescence wavelength shift, anisotropy, lifetime, and energy transfer have all been reported for designing aptamer-based sensors. 5 Most fluorescent aptamer sensors involve cuvette-based measurements, in which the sensor and target analyte are mixed in a cuvette and the change of fluorescence signal is monitored with a fluorometer.…”

“…21,28 On the other hand, at least 100-500 L of sample is needed to fill a fluorescence cuvette. For practical application, it is desirable to decrease the sample volume to a level comparable to that used for glucose detection (e.g.…”