A) Serum sample collection
1. Filled red-top blood-collection tubes should be kept upright for 30-60 min after the blood was drawn at room temperature to allow the clot to form.
!CAUTION All experiments involving humanss would be performed according to institutional and governmental guidelines.
▲CRITICAL STEP Use red-top (serum) silicon-coated tubes with no additives, rather than serum separator tubes. These tubes, without additives, allow the red blood cells to form a clot, which also includes white blood cells and platelets, and which can be separated from the serum by centrifuging. The red-top tubes do not have to be full to be used.
2. Centrifuge the blood sample after clotting for 30–60 min using a horizontal rotor (swing-out head) for 20 min at 1100–1300 × g at room temperature. If the blood is not to be centrifuged immediately after clotting, the tubes should be refrigerated at 4 ºC for no longer than 4 h.
!CAUTION Excessive centrifuge speed (over 2000 × g) may cause tube breakage, exposure to blood serum, and possible injury.
3. Use a pipette to transfer the serum. If more than one tube is drawn per sample, pull the serum from both tubes into a 15 mL conical tube and mix. Pipette the serum into labeled cryovials and close the caps tightly. Place all aliquots upright in a specimen box or rack at 4 °C
▲CRITICAL STEP Do not pour the serum. This process should be completed within 1 h of centrifugation. Take care not to pick up red blood cells when aliquoting by keeping the pipette above the red blood cell layer and leaving a small amount of serum in the tube. Check that all aliquot vial caps are secure and that all vials are labeled.
■PAUSE POINT The serum samples can be stored at 4 °C for up to 1 month.
B) Preparation of peptide 1 and probe 1
4. The Rink amide resin (312 mg, 0.8 mmol/g, 0.25 mmol, 1.0 equiv.) was weighed out into a plastic peptide-synthesis vessel and allowed to swell in DCM (10.0 mL) for 1.0 h.
5. The Rink amide resin was washed by an intensive cycle of DMF( 5×10 mL, 2 min each) and the following amino acids [Fmoc-Arg(Pbf)-OH; Fmoc-Lys(Boc)-OH; Fmoc-Lys(Alloc)-OH; Fmoc-Gln(Trt)-OH; Fmoc-Glu(OtBu)-OH; Fmoc-Phe-OH; Fmoc-Met-OH; Fmoc-Leu-OH; Fmoc-Gly-OH; Fmoc-Pro-OH] were attached under microwave radiation conditions (35 W, 60 ± 5 °C, 20 min) according to a standard protocol: Fmoc-AA-OH (0.75 mmol, 3.0 equiv.), PyBOP (0.75 mmol, 3.0 equiv.), and DIPEA (1.5 mmol, 6 equiv.) in DMF (10.0 mL).
6. The Fmoc protection group was removed by agitation with 8.0 mL of 20 % piperidine/DMF (vol/vol) under microwave radiation conditions (35 W, 60 ± 5 °C, 30 s), and the solvent was then removed by vacuum filtration. Second, the Fmoc protection group was removed by agitation with 8.0 mL of 20 % piperidine/DMF (vol/vol) under microwave radiation conditions (35 W, 60 ± 5 °C, 5.0 min), and the solvent was then removed by vacuum filtration.
7. After Fmoc-Lys(Alloc)-OH was coupled, the Alloc group was removed by adding Pd(PPh3)4 (0.05 mmol, 0.2 equiv.) and PhSiH3 (12 mmol, 48 equiv.) in DCM (10 mL) under argon for 30 min. Terpyridine (1.5 mmol, 6.0 equiv.), 2-(4-((2-carboxyethyl)(ethyl)amino)styryl)-1,1,3-trimethyl-1H-benzo[e]indol-3-ium (0.75 mmol, 3.0 equiv.), and 2-(4,7,10-tris(2-(tert-butoxy)-2-oxoethyl)-1,4,7,10-tetraazacyclodecan-1-yl) acetic acid (0.75 mmol, 3.0 equiv.) were then coupled at room temperature.
8. The product was cleaved from Rink amide resin using a cleavage cocktail of TFA-Tis-H2O (95:2.5:2.5, 10 mL) for 3 h.
9. TFA was removed and the peptide was precipitated three times by cold diethyl ether and centrifuged at speed (1000 × g) for 8 min at room temperature.
Purification and preparation of peptide 1
10. The crude peptides were purified by high-performance liquid chromatography on an RP18-column using water/CH3CN (with 0.05 % TFA) as the elution buffer.
11. The solid was dissolved in water (20 mL) acidified with hydrochloric acid (0.1 N, 5 mL) and lyophilized. This step was repeated three times. The product was identified by MALDI-TOF.
12. Complexation of the ligand with Gd (III)-ion was performed by the dropwise addition of an aqueous solution of GdCl3 to an aqueous solution of the ligand at pH 3.0. The mixture was then stirred for 6 h, and the pH was adjusted to 6.5 using a 1M solution of NaOH. The final Gd (III)-peptide 1 complex was obtained as a purple powder after lyophilization (excess GdCl3 was dialysed in MilliQ grade water). The formation of the complex was identified by MALDI-TOF.
13. Peptide 1 was dissolved in TBS buffer (pH 7.4, 50.0 mM Tris, 50.0 mM NaCl) at a concentration of 1 mM.
!CAUTION The powder of peptide 1 may be inhaled. Appropriate personal protective clothing should be worn during the experiment and the preparation and use should be carried out in a clean bench to avoid microbial contamination. All solutions must be filtered prior to use.
■PAUSE POINT The peptide 1 solution should be stored at 4 °C and can remain stable for at least 1 month.
Preparation of probe 1
14. Add the same eq. of FeCl2 to the peptide 1 solution. The final probe 1 was obtained as a purple powder after lyophilization (excess FeCl2 was dialysed in MilliQ grade water).
!CAUTION The FeCl2 solution must be filtered prior to use. It is strongly recommended to perform the experiment at 4 °C, because Fe2+ is oxidized by air at higher temperatures.
15. Incubate the mixture at 4 °C for at least 24 h to allow formation of the Fe-peptide 1 complex and further aggregation into probe 1.
■PAUSE POINT The peptide 1 solution should be stored at 4 °C and may remain stable for at least 1 month.
▲CRITICAL STEP Although the process of Fe-peptide 1 complex formation takes < 1.0 min, the aggregation process takes longer. The mixture should thus be incubated for at least 24 h to allow the Fe(II)-peptide 1 complex to aggregate into probe 1.