Silk-NH2 lyophilized protein was resuspended in phosphate buffered saline with 5mM EDTA, pH 8.0 (PBS/EDTA). To 1.16 mL Silk-NH2 solution, 8 mg of sulfo-LC-SPDP (sulfosuccinimidyl 6-[3′-(2-pyridyldithio)propionamido]hexanoate) (Thermo Fisher, Rockford, IL) was dissolved and incubated at room temperature for 1 hour to link the sulfo-LC-SPDP to the free amines, creating Silk-LC-SPDP. The Silk-LC-SPDP solution (80 μl, or 4.8 mg silk) was then cast onto PDMS cylinders and allowed to dry into films overnight. Films were washed 3x with PBS for 20 minutes each to remove residual sulfo-LC-SPDP. Successful conjugation of sulfo-LC-SPDP to the Silk-LC-SPDP films was evaluated by measuring the release of the pyridine-2-thione ring of the sulfo-LC-SPDP after treatment with 15 mg/mL dithiothreitol (DTT) for 30 min and measuring the absorbance at 343 nm, as per the manufacturer’s directions.
Sulfo lc spdp
Manufactured by Thermo Fisher Scientific
Sourced in United States
Sulfo-LC-SPDP is a heterobifunctional crosslinking reagent that contains an N-hydroxysulfosuccinimide (NHS) ester on one end and a pyridyl disulfide group on the other end. It is used for the covalent conjugation of proteins, peptides, or other biomolecules containing primary amines with those containing free sulfhydryl groups.
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Lab products found in correlation
Sulfo smcc, by Thermo Fisher Scientific (2 mentions)
Polyethylene glycol, by Merck Group (1 mentions)
Lactate dehydrogenase ldh, by Merck Group (1 mentions)
5 iodoacetamidofluorescein 5 iaf, by Thermo Fisher Scientific (1 mentions)
β nicotinamide adenine dinucleotide, by Merck Group (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Merck Group (1 mentions)
Glutaraldehyde, by Merck Group (1 mentions)
Sodium pyruvate, by Merck Group (1 mentions)
Goat anti rabbit igg h l alexa fluor 488, by Abcam (1 mentions)
Horse cyt c, by Merck Group (1 mentions)
Mrc 5, by American Type Culture Collection (1 mentions)
Alexa fluor 488 conjugated goat anti rabbit secondary antibody, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 568 conjugated phalloidin, by Thermo Fisher Scientific (1 mentions)
Fura 2 acetoxymethyl ester, by Thermo Fisher Scientific (1 mentions)
Lyophilized fibrinogen from human plasma, by Merck Group (1 mentions)
ε aminocaproic acid εaca, by Merck Group (1 mentions)
Insulin transferrin sodium selenite media supplement, by Merck Group (1 mentions)
Millex syringe filter, by Merck Group (1 mentions)
Whatman syringe filter, by GE Healthcare (1 mentions)
Hydrocortisone, by Merck Group (1 mentions)
12 protocols using sulfo lc spdp
1
Silk-LC-SPDP Conjugation Protocol
2
Antibody Immobilization on Gold Electrodes
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The polyclonal anti-Candida albicans antibody were acquired from Abcam (ab53891, Cambridge, UK). Functionalization of the gold electrodes with antibodies was performed following a modified protocol described in [19 (link)]. The first step requires modification of the antibodies with a crosslinker by incubating 2 mg/mL antibody solution with freshly prepared 20 mM solution of sulfo-LC-SPDP (sulfosuccinimidyl 6-[3’(2-pyridyldithio)-propionamido]hexanoate) (Thermo Scientific, Waltham, MA, USA) for 1 h at room temperature. After the incubation the reaction by-products and unreacted sulfo-LC-SPDP are removed by means of desalting column (Zeba Spin, Thermo Scientific). Once the antibodies are modified with disulfides a 100 g/mL solution is prepared and incubated overnight with the membrane electrodes.
3
Sensitive Protein Detection Protocol
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The following materials were used in this study: sulfo-LC-SPDP (sulfosuccinimidyl 6-(3′-(2-pyridyldithio)propionamido)hexanoate; ThermoScientific, Waltham, MA, USA, 21650), TCEP-HC (Tris(2-carboxyethyl)phosphine hydrochloride Thermo Scientific, Waltham, MA, USA, 20490), EDTA (ethylenediaminetetraacetic acid; Sigma Aldrich, Burlington, MA, USA, E9884), MicroDeckTM-G-150 surfaces (Bioactive Surfaces, Galapagar, Madrid, Spain), lactate dehydrogenase (LDH; Sigma Aldrich, Burlington, MA, USA, L1006-12.5KU), biotinylated anti-lactate dehydrogenase (anti-LDH) antibodies (IgG polyclonal antibody; Origene, Rockville, MD, USA, AP21329BT-N), poly(ethylene glycol)-(N-hydroxysuccinimide 5-pentanoate) ether 2-(biotinylamino)ethane (NHS–PEG–biotin; Sigma Aldrich, Burlington, MA, USA, 757799-100μγ), streptavidin (Sigma Aldrich, Burlington, MA, USA, S4762-5 mg), DeepTipTM SiN R11 atomic-force microscopy probes (Bioactive Surfaces, Galapagar, Madrid, Spain), Goat Anti-Rabbit IgG H&L Alexa Fluor® 488 (Abcam, Cambridge, UK, ab150077), 5-iodoacetamidofluorescein (5-IAF; Thermo Scientific, Waltham, MA, USA, 62246), sodium pyruvate (P2256 Sigma-Aldrich, Burlington, MA, USA,), β-nicotinamide adenine dinucleotide (NADH; N8129-500 mg), and glutaraldehyde (50 wt. % in H2O, Sigma Aldrich, Burlington, MA, USA, 340855).
4
Biomarker Quantification Using Gold-SPE
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Biomarker quantification was performed using gold screen-printed electrodes (SPEs) (DropSens, C223AT) with a printed gold working electrode (WE) (1.6 mm ∅), a gold counter electrode (CE), and a silver pseudo reference electrode (RE) (Cruz et al., 2019 (link), 2021 ). The gold-SPEs were pre-cleaned with isopropanol and deionized (DI) water, prior to functionalization. To form the self-assembled monolayer (SAM) the sensor was incubated for 20 min at room temperature (RT), with a solution (10 mg/mL) of sulfo-LC-SPDP (sulfosuccinimidyl 6-(3′-(2-pyridyldithio)propionamido)hexanoate) (Thermo Fisher) in 10 mM phosphate buffer (PB, pH7.4) with 5% glycerol and washed with PB. Anti-IL-6 antibodies (eBioscience#88-7066) (0.25 μg/μL antibody solution (in PB) with 5% glycerol), were linked to SAM through overnight incubation, at 4 °C, followed by rinsed with PB. Next sensors were incubated with 0.25% BSA solution in PB at RT for 30 min with 5% glycerol to block non-specific interactions and unreacted sites.
5
Functionalization of Spin-Valve Biosensor
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The MR biochip is composed of an array of 30 U-shaped spin-valve (SV) sensors and was microfabricated, as reported previously [30 (link),31 (link),32 (link)]. The gold thin layer (12.9 × 35.4 μm2) above the SV sensors was treated with a heterobifunctional surface linker: 1 mg/mL of sulfosuccinimidyl 6-[3′-(2-pyridyldithio)propionamido] hexanoate (sulfo-LC-SPDP, Thermo Scientific, Waltham, MA, USA) in a PB for 20 min. Then, 1 µL of the monoclonal antibodies (Abcam, Cambridge, UK) at 250 µL/mL, made contact with the sensor surface of 15 SV sensors for 2 h at room temperature (RT), followed by a blocking step with 1% of the BSA. The remaining 15 SV sensors were used as negative control sensors and contacted with 5% of the BSA.
6
Protein Labeling and Characterization
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Sulfosuccinimidyl 6-(3'-(2-pyridyldithio)propionamido)hexanoate (Sulfo-LC-SPDP) and Sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (Sulfo-SMCC) were purchased from Thermo Scientific (Rockford, IL, USA). A549, HeLa, K562, and MRC-5 cells were obtained from ATCC (Manassas, VA, USA). Human serum Apo-Tf, horse Cyt c, and other buffers and salts were obtained from Sigma-Aldrich (St. Louis, MO, USA). Apo-Tf was further processed to obtain the iron bound form of Tf and the amount of bound iron was determined by the ferrozine assay as described [27 (link)]. Apo-Tf concentration was calculated by using the molar absorption coefficient of 93 × 103 M-1 cm-1 at 278 nm [28 (link)]. The molar ratio of 2:1 for bound iron to Tf was used to confirm that both Tf iron binding sites were occupied.
7
Bioconjugation of Antibodies to Plasmonic Nanoparticles
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Bioconjugation of antibodies to plasmonic nanoparticles was carried out with the heterobifunctional crosslinker sulfosuccinimidyl 6-[3′-(2-pyridyldithio)-propionamido] hexanoate (Sulfo-LC-SPDP, 21650, Thermo)67 (link),68 . A total of 100 µM Sulfo-LC-SPDP was prepared in ultrapure water immediately before use. Then, 25 µL of Sulfo-LC-SPDP were added to 10 μg IgG dissolved in 0.5 mL of PBS-EDTA (100 mM sodium phosphate, 150 mM NaCl, 1 mM EDTA, 0.02% sodium azide, pH 7.5) and incubated for 30 min at room temperature (RT). Conjugated antibodies were exchanged with 40 mM HEPES buffer using a 10 K MWCO centrifugal filter (Millipore). Plasmonic nanoparticles (40 nm GNPs and 40 nm SNPs) were centrifuged (3000×g, 15 min) and resuspended in 40 mM HEPES buffer before antibody conjugation. Plasmonic nanoparticles and antibodies were mixed at a 1:250 molar ratio and incubated overnight at 4 °C.
8
Fibrinogen Hydrogel Tissue Engineering
9
Synthesis of Maleimide-Activated Glycopeptide
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HGVTSAPD (Neu5Acα2-3Galβ1-3GalNAc) TRPAPGSTAPPA (PDT*R-23ST-20-mer)
was modified by sulfo-SMCC (Thermo Fisher Scientific) to make maleimide-activated
glycopeptide. The sulfhydryl groups were introduced to BSA by the
reaction with sulfo-LC-SPDP (Thermo Fisher Scientific). The sulfhydryl-activated
BSA was incubated with the maleimide-activated glycopeptide. The reaction
mixture was dialyzed against distilled water and then freeze-dried
(immunogen-1:Figure 1 ).
was modified by sulfo-SMCC (Thermo Fisher Scientific) to make maleimide-activated
glycopeptide. The sulfhydryl groups were introduced to BSA by the
reaction with sulfo-LC-SPDP (Thermo Fisher Scientific). The sulfhydryl-activated
BSA was incubated with the maleimide-activated glycopeptide. The reaction
mixture was dialyzed against distilled water and then freeze-dried
(immunogen-1:
10
Sialidase-Based Targeted Therapy Complex
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Fifty milligrams of sialidase (90 kDa) was weighed and dissolved in PBS-EDTA solution, and the free carboxyl group was calculated according to the amino acid sequence. Then, a specific weight of Sulfo-LC-SPDP (Thermo Scientific) powder was weighed and added to the solution according to the molar ratio of the carboxyl group at 1:10. After the solution was stirred in the dark and reacted at 16 °C overnight, Millipore Merck Amicon® Ultra15 10-kDa ultrafiltration centrifuge tubes were used for segregation at 3,500 × g for 20 min to remove any unreacted Sulfo-LC-SPDP Linker. Next, an accurate quantity of non-small cell lung cancer-TP (CTDSILRSYDWTY) was weighed and added to the solution at a molar ratio of 1:10, and the mixture was magnetically stirred at 16 °C for 24 h to allow coupling. Then, the mixture was centrifuged with an ultrafiltration tube to remove any unconnected TP. Finally, the obtained targeting complex molecules were lyophilized, and NMR and infrared spectroscopy (IR) were employed to determine whether the complex molecules (TP-Sia) were successfully synthesized. Before the complex molecules (TP-Sia) were used, FITC-modified SNA was also employed to detect the enzymatic activity.
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