Förster Resonance Energy Transfer-Based Self-Assembled Nanoprobe for Rapid and Sensitive Detection of Postoperative Pancreatic Fistula

Abstract

Postoperative pancreatic fistula (POPF) is the most serious and challenging complication following gastroenterological surgery. Activated pancreatic juice leaking from the organ remnant contains proteases that attack the surrounding tissue, potentially leading to severe inflammation, tissue necrosis, and fistula formation. However, it is difficult to observe pancreatic leakage during surgery and to evaluate the protease activity of leaked fluid at the patient's bedside. This report describes a protein nanocage-based protease ratiometric sensor comprising a pancreatic protease-sensitive small heat-shock protein (HSP) 16.5, which is a naturally occurring protein in Methanococcus jannaschii that forms a spherical structure by self-assembly of 24 subunits, and a chemically conjugated donor-acceptor dye pair for Förster resonance energy transfer (FRET). The HSP-FRET probe was constructed by subunit exchange of each dye-labeled engineered HSP, resulting in a spherical nanocage of approximately 10 nm in diameter, which exhibited very high stability against degradation in blood plasma and no remarkable toxicity in mice. The efficiency of FRET was found to depend on both the dye orientation and the acceptor/donor ratio. Pancreatic proteases, including trypsin, α-chymotrypsin, and elastase, were quantitatively analyzed by fluorescence recovery with high specificity using the HSP-FRET nanoprobe. Furthermore, the HSP-FRET nanoprobe was sufficiently sensitive to detect POPF in the pancreatic juice of patients using only the naked eye within 10 min. Thus, this novel nanoprobe is proposed as an effective and convenient tool for the detection of POPF and the visualization of activated pancreatic juice during gastroenterological surgery.

SmartPAN: A novel polysaccharide-microsphere-based surgical indicator of pancreatic leakage

“…These properties make the technique unattractive for routine usage. Finally, another team from Kyushu University 17 devised a Förster resonance energy transfer (FRET) heat-shock protein probe. Again, excitation of the probe and detection of the FRET signal require specialist equipment not readily available in most operating theatres.…”

“…At present, there are no reliable ways to predict POPF, take prophylactic peri-operative measurements or perform targeted closure during surgery. 16 Some attempts have been made to develop a method for intraoperative identification of pancreatic fluid leakage, [17][18][19][20][21] but none are clinically useful. The ideal indicator of pancreatic fluid leakage would show high sensitivity and specificity in situ with a clear and discernible reaction.…”

Liposome-based Biosensors and Diagnosis Imaging Agents

“…It is also a sensitive tool for detecting molecular binding and changes in protein conformation, as well as for chemical and biological analyses. (41)(42)(43) Liposomes have a bilayer structure and a thickness of approximately 5 nm; accordingly, FRET is highly suitable for biosensor applications. Moreover, a liposomal nanoplatform reported by Wan et al encapsulated upconversion nanoparticle (UCNP)-gold nanoparticle (AuNP) nanocomplexes, which could measure ROS concentrations via FRET imaging while also scavenging excessive ROS.…”

A Surface Plasmon Resonance Plastic Optical Fiber Biosensor for the Detection of Pancreatic Amylase in Surgically-Placed Drain Effluent

“…In general, the detection of amylases by means of biosensors are mostly based on electrochemical transduction systems and, in particular, are focused on the detection of amylases in saliva [ 6 , 7 , 8 , 9 , 10 ] and in human serum [ 11 , 12 ]. Concerning the measurement of amylase in drain fluids, to date just a single example of a biosensor is reported [ 13 ]. In this work, Förster resonance energy transfer (FRET) was selected as the working principle for the sensor and a labelled protein-assembly nanoprobe, sensitive to activated pancreatic proteases (i.e., elastase, α-chymostrypsin, trypsin), was able to optically detect activated pancreatic juices.…”