Abstract
Herein, a fluorescence turn-on nanosensor (MnIO@pep-FITC) has been proposed for detecting trypsin activity in vitro and in vivo through covalently immobilizing an FITC modified peptide substrate of trypsin (pep-FITC) on manganese-doped iron oxide nanoparticle (MnIO NP) surfaces via a polyethylene glycol (PEG) crosslinker. The conjugation of pep-FITC with MnIO NPs results in the quenching of FITC fluorescence. After trypsin cleavage, the FITC moiety is released from the MnIO NP surface, leading to a remarkable recovery of FITC fluorescence signal. Under the optimum experimental conditions, the recovery ratio of FITC fluorescence intensity is linearly dependent on the trypsin concentration in the range of 2 to 100 ng mL−1 in buffer and intracellular trypsin in the lysate of 5 × 102 to 1 × 104 HCT116 cells per mL, respectively. The detection limit of trypsin is 0.6 ng mL−1 in buffer or 359 cells per mL HCT116 cell lysate. The MnIO@pep-FITC is successfully employed to noninvasively monitor trypsin activity in the ultrasmall (ca. 4.9 mm3 in volume) BALB/c nude mouse-bearing HCT116 tumor by in vivo fluorescence imaging with external magnetic field assistance, demonstrating that it has excellent practicability.
Highlights
As paradigms of enzyme catalysis, serine (Ser) proteases play critical roles in a variety of biological and physiological processes such as cell differentiation, cell apoptosis, blood coagulation, atherosclerosis, in ammation and cancer.[1,2,3,4,5,6] The Ser proteases normally contain a nucleophilic Ser residue at their active site, which has nucleophilic activity against the peptide bond resulting in the digestion of some proteins implicated in vital activities
Peptide substrate-based biosensors have been considered as a promising way to determine trypsin activity in different matrixes since peptide substrates exhibit several unique merits, such as easy synthesis, low cost, high stability in harsh environments and suitable to conjugate/modify with other label molecules.[33,34,35,36,37,38]
The hydrophobic OA capped manganese-doped iron oxide nanoparticle (MnIO NP) were transferred into aqueous phase through ligand exchange of OA with DIB-polyethylene glycol (PEG)-COOH by formation of Mn(II)/Fe(III)-DIB complex.[42]
Summary
A large number of methods/assays have been developed for quanti cation of trypsin levels in vitro including chromatography,[17] radioimmunoassay[18] and biosensors with different detection principles.[19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38] Among of these techniques, peptide substrate-based biosensors have been considered as a promising way to determine trypsin activity in different matrixes since peptide substrates exhibit several unique merits, such as easy synthesis, low cost, high stability in harsh environments and suitable to conjugate/modify with other label molecules.[33,34,35,36,37,38] For example, a variety of peptide-based uorescence turn-on/off strategies have been developed for sensing trypsin activity with high sensitivity and selectivity.[32,33,34] most of asdeveloped sensing platforms are only capable of trypsin activity detection in vitro, which does not necessarily re ect the quantitative information on trypsin expression in a solid tumor. Paper a challenge to develop peptide-based biosensors for measurement of tumor-related trypsin activity in vivo since it is difficult to efficiently transport peptide substrate to tumor site. A peptide-functionalized manganese-doped iron oxide nanoparticle-based uorescence turn-on sensing platform (MnIO@pep-FITC) has been constructed for noninvasive detection of trypsin activity in vivo through conjugation of FITC modi ed peptide substrate on the MnIO NP surface. A er incubated for 2 h, the activated MnIO@PEG were collected by centrifugation (6000 rpm, 10 min), and reacted with pep-FITC (0.1 mg mLÀ1 in 10 mL PBS (10 mmol LÀ1, pH 7.4)) under stirring for another 24 h. 100 mg mLÀ1 MnIO@pep-FITC were incubated with various concentrations of trypsin in 500 mL PBS (10 mmol LÀ1, pH 7.4) at 37 C for 90 min. MnIO@pep-FITC were incubated with 100 ng mLÀ1 a series of enzymes including matrix metalloproteinases (MMP-2, MMP-7 and MMP-9), caspase-3 and caspase-9 under optimized conditions
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