Traut s reagent

First, 2 mg of ntPE-TEV-H6 protein was reacted with TEV enzyme (Life Technologies, Carlsbad, CA, USA) and a 1 mL HisTrap HP column was used to elute the cleaved protein (ntPE-CENLYFQ). A 5-fold molar excess of N-(ε-maleimidocaproic acid) hydrazide (EMCH) was reacted with ntPE at RT for 2 h prior to the removal of unreacted EMCH using dialysis (10 kDa MWCO) against PBS overnight at 4 °C. To prepare a control protein for coupling to NPs, a 3-fold molar excess of Traut’s reagent (5,5′-dithiobis(2-nitrobenzoic acid); Sigma-Aldrich, Gillinghamm, UK) was added to BSA in 12 mM PBS (pH 7.4) containing 3 mM EDTA and incubated at RT for 1 h to produce ~1 thiol group per BSA molecule prior to reaction with EMCH in a manner identical to that used for ntPE-CENLYFQ.

DFS requires the strong immobilization of the molecules at the cantilever probe for them not to unbind while withdrawing in force scans. Prefunctionalized maleimide-terminated flexible polyethyleneglycol (PEG) linker silicon nitride AFM cantilevers (MW 3400; Novascan Technologies Inc., Ames, IA, USA) were used. Nominal stiffness constant values of cantilevers ranged from 0.02 to 0.06 N/m. For NADP⁺ functionalization, a 2 mg/mL 2-iminethiolane solution (Traut’s Reagent; Thermo Scientific Pierce, Waltham, MA, USA) in 5 mM PBS/EDTA pH 7.2 was prepared. NADP⁺ (Sigma-Aldrich, San Luis, MO, USA) and Traut’s Reagent solution were mixed in a 1:10 molar ratio and incubated for 2 h at RT in the absence of light (Figure 3). Then, 150 μL of the aforementioned NADP⁺ mixture was added to the maleimide–PEG cantilever fixed on a capsule, and incubated overnight at 4 °C in the absence of light (Figure 3). AFM tips were washed three times (5 min each) with the same buffer and kept at 4 °C in the absence of light until use.

The HDL-mimetic peptide-lipid nanoparticles (HPPS) which were modified by DSPE-PEG (2000) maleimide around its surface were prepared and purified as reported before [9 (link),10 (link)]. 27μl Traut’s reagent (25 mg/ml, Sigma Aldrich, USA) was mixed with 360μl anti-TfR mAb (10 mg/ml) in PBS. The mAb-SH could not be collected until 1h reaction of the mixture under a roller mixer at room temperature. Then a Ultrafiltration device (Amicon Ultra-15, 30000 MWCO, MERK) was used to remove the excess Traut’s reagent. The anti-hTfR mAb conjugated HPPS (HPPS-mAb) was prepared by mixing mAb-SH with maleimide-containing HPPS and shaking at room temperature for 20h. The nanostructure carried DIR-BOA, a near-infrared fluorescent dye as cargo. Concentration of DIR-BOA was determined from a standard curve of DIR-BOA according to the same protocol as described before [10 (link),11 (link)].

Hexaamineruthenium (III) chloride (Ru(NH₃)₆³⁺), methylene blue, phosphate-buffered saline (PBS, pH 7.4), Traut’s Reagent, (ethylenedinitrilo)tetraacetic acid (EDTA), ascorbate oxidase (AO), casein, and other reagents for buffer solutions were purchased from Sigma-Aldrich (St. Louis, MO). Deionized (DI) water (18.3 MΩ-cm⁻¹) was generated using a Smart2Pure water purification system. StabilBlock® Immunoassay Stabilizer was purchased from SurModics, Inc. (Eden Prairie, MN). Mouse monoclonal anti-PfHRP2 IgM and mouse monoclonal anti-PfHRP2 IgG were purchased from ICL, Inc. (Portland, OR). Recombinant P. falciparum histidine-rich protein 2 (PfHRP2) and P. falciparum lactate dehydrogenase (PfLDH) were purchased from CTK Biotech (San Diego, CA).