Standard-free single magnetic bead evaluation: a stable nanoplatform for prostate disease differentiation

Explicit interpretation of heterogeneity between prostate-specific antigen (PSA) subtypes is essential for prostate cancer differentiation during different disease courses, whereas a universal protocol with uniform criteria is still lacking across the globe. In this work, a standard-free single magnetic bead (SMB) nanoplatform utilizing metal nanoparticles with optimal diameters was proposed for prostate disease differentiation in a 134-donor model. The inaccuracy of detection in absolute quantification was diminished via evaluations of metal intensities on the single magnetic bead. The intrinsic proportion of fPSA in tPSA was successfully evaluated by direct use of the Pt to Au intensity ratio (Pt/Au ratio), exhibiting better differentiation between healthy and unhealthy, benign prostatic hyperplasia (BPH) and cancer individuals compared with solo fPSA or tPSA. We generated thresholds respectively for prostate disease differentiation, envisioning that this standard-free SMB nanoplatform would establish a standardized methodology with uniform criteria worldwide in cancer diagnosis, staging, and postoperative assessments.


Apparatus
Absorption of AuNPs and PtNPs with three different diameters were measured by Cytation 5 Cell Imaging Multi-Mode Reader (BioTek Instruments, Inc.) using polystyrene 96-well microtiter plates (Jet Bio-Filtration Co., Ltd.). Diameters of nanoparticles were implemented by Malvern Nano ZS90 (Malvern Panalytical Ltd.). NexION-350 inductively coupled plasma mass spectrometer (PerkinElmer, Inc.) with single particle mode was applied for data collecting in SMB nanoplatform. Working conditions of SMB nanoplatform were listed in Table  S1. Transmission electron microscopy (TEM) images were obtained by a JEM-2010 microscope (JEOL Co., Japan) and diameters of nanoparticles were calculated using ImageJ (1.52a version).

Synthesis and Labeling of Nanoparticle Probes
A series of AuNPs with different diameters were synthesized according to former articles with slight modifications. 1, 2 Briefly, 1.1 mL 1% trisodium citrate was added in a three-necked flask with 75 mL ultra-pure water. After boiling for 10 min, HAuCl 4 (0.01%, w/v) was added and kept refluxing for 20 min. Then the solution was cool down to 90 o C, followed by adding 0.5 mL HAuCl 4 (0.01%, w/v) into the solution again. The procedure was repeated one more time to obtain 21 nm AuNPs. 31 nm AuNPs were synthesized by repeating the above procedure three times. 47 nm AuNPs were synthesized by one-step reduction where 1 mL 1% trisodium citrate was added to boiling solution containing 100 mL ultrapure water and 0.01% HAuCl 4 . The synthesis of PtNPs with three different diameters was carried out based on the seed growth method. 3 Firstly, 5 nm Pt seeds were synthesized by successively adding a mixture of 1% trisodium citrate and 0.05% citrate acid, 0.55 mL 1% trisodium citrate, and 0.1% KBH 4 to 50 mL boiling solution containing 0.2% chloroplatinic acid. Secondly, 1% chloroplatinic acid and 1.25% ascorbic acid was mixed with 2 mL 5 nm Pt seeds and ultrapure water in a three-necked flask, followed by boiling and refluxing to obtain 18 nm PtNPs. 36 nm and 51 nm PtNPs were synthesized respectively by adding 11 mL and 8 mL 18 nm PtNPs in a three-necked flask together with ultrapure water, 1% chloroplatinic acid, and trisodium citrate/ascorbic acid mixture. All as-synthesized nanoparticles were purified by centrifuge and characterized by UV, TEM, DLS, and sp-ICP-MS.

Establishment of SMB Nanoplatform
Magnetic bead-centered SMB nanoplatform was established by preparing antibody labeled magnetic beads. Briefly, 20 µL 10mg/mL carboxylic acid-coated magnetic beads were firstly activated by EDC (150 mg/mL) and sulfo-NHS (25 mg/mL) under the 6.5 pH condition. MES solved coating-anti-tPSA antibody was then mixed with magnetic beads. Coupling between carboxyl on magnetic beads and amino groups on antibodies took 12 hours. Antibody labeled magnetic beads were finally blocked by 10% BSA-contained PBS buffer and washed three times by 1% BSA-contained PBS buffer. Concentrations of both AuNPs and PtNPs with three different diameters were counted by the digestion method mentioned in our previous work. 4 Nanoparticles were digested by aqua regia (1HNO 3 :3HCl, v/v) and ions concentrations were subsequently measured by standard mode (Shown in Fig. S6). After adjusting the nanoparticle concentrations to an appropriate range, nanoparticle probes were synthesized by electrostatic attachments. Primarily, 1 mL boric acid buffer dispersed AuNPs (pH 8.2) were mixed with 30 µL 1 mg/mL labeling-anti-tPSA antibody which was optimal as the best volume according to our previous work. 4 The attachment took 1 hour, followed by another surface blocking hour by 10% BSA PBS buffer. Antibody labeled AuNPs were purified from excessive antibody and BSA by 16 min centrifugation. 500 µL 1% BSA PBS buffer was applied to redisperse the nanoparticles after discarding the supernatant. The scenario of PtNPs labeling was carried in the similar way. Mixed probes were synthesized by adding as-prepared 500 µL AuNPs probes and 500 µL PtNPs probes into a 1.5 mL centrifugal tube.

Simultaneous Evaluation of tPSA and fPSA
Under the fact that fPSA is a subtype of tPSA which is not bound with protease inhibitors, 5 standard curves of fPSA and tPSA were implemented separately in this work. The standard curve of fPSA was implemented by mixing varied concentrations of fPSA with mixed probes, while the standard curve of tPSA was carried out by mixing tPSA with mixed probes. Simultaneous detection of tPSA and fPSA in serum samples by SMB nanoplatform was implemented as followed: 5 µg antibody labeled magnetic beads were added in a 200 µL PCR tube, followed by adding 60 µL diluted serum samples and 25 µL mixed probes. All serum sample was diluted at least two times to reduce matrix effects. Samples were incubated under 37 o C for 2 hours, followed by two times washing using 1% BSA PBS buffer containing 0.05-0.1% Tween-20. Before SMB detection, samples were diluted 400 times using ultrapure water. In sp-ICP-MS mode, 58 Fe + was applied for magnetic beads counting, making all measurements under the same number of magnetic beads. 194 Pt + and 197 Au + were chosen for evaluating Pt and Au intensity on each magnetic bead.

Selectivity and Stability
Selectivity of the proposed SMB nanoplatform was carried out by mixing nanoparticle probes and magnetic beads with tPSA/fPSA (50 ng/mL for both) or interferences (500 ng/mL for IgG, 500 ng/mL for AFP, 500 ng/mL for CEA, 500 U/mL for CA199, 500 U/mL for CA125 and 500 U/mL for CA153). Measurements of Pt and Au were carried out simultaneously by sp-ICP-MS. The stability of the SMB nanoplatform was explored by successively applying immunoassay using 50 ng/mL fPSA and 50 ng/mL tPSA.

Statistical Analysis
In this article, the average intensity of Au or Pt per magnetic bead was calculated using the formula:

Fig. S1: Transient 58 Fe + Signals of Magnetic Beads in sp-ICP-MS
Magnetic beads counting was carried out using 58 Fe isotopes in sp-ICP-MS. Each pulse above the threshold (10 counts) represents a detected magnetic bead.

Fig. S2: Identification and Differentiation of AuNPs and PtNPs by Appearances and TEM Elemental Mapping
Due to the different appearances, AuNP and PtNPs were identified and differentiated by roughness on the surface (a). PtNPs were synthesized by seed growth method where crystallinity and roughness existed on the surface, while the surface of AuNPs was smooth.
The captured AuNPs and PtNPs on a single magnetic bead were also observed by TEM mapping (b).

Fig. S3: Characterizations of PtNPs with Three Different Diameters
In this work, TEM was applied for getting the diameters of PtNPs. ImageJ (1.52a version) was used for the diameter analysis where the ruler in TEM image was considered as the standard. Generally, 18±2 nm, 36±2 nm and 51±3 nm PtNPs were synthesized for diameter-regulation immunoassays. DLS and sp-ICP-MS were also applied respectively for characterizations.

Fig. S4: Characterizations of AuNPs with Three Different Diameters
In this work, TEM was applied for getting the diameters of AuNPs. ImageJ (1.52a version) was used for the diameter analysis where the ruler in TEM image was considered as the standard. Generally, 21±3 nm, 31±3 nm and 47±3 nm AuNPs were synthesized for diameter-regulation immunoassays. DLS and sp-ICP-MS were also applied respectively for characterizations.

Fig. S5: UV Spectrometry of AuNPs and PtNPs
UV was utilized to depict the peak of absorbance. The peaks of AuNPs were found as 520 nm for 21 nm, 528 nm for 31 nm, and 534 nm for 47 nm. The peaks of PtNPs were found as 237 nm for 18 nm, 251 nm for 36 nm, and 306 nm for 51 nm.

Fig. S6: Concentration Measurements of AuNPs and PtNPs
External standard method was applied for measuring the ion concentrations of AuNPs and PtNPs after digestion by aqua regia. Concentrations of nanoparticles were calculated using formula (detailed nanoparticle concentrations were shown in Table S3): Where was number concentration of nanoparticles, was ion concentration of Au of Pt after digestion by aqua regia. represented the diameter of nanoparticle. N A represented the Avogadro constant.

Fig. S7: Relationship Between Nanoparticle Diameter and Metal Isotopic Intensity in sp-ICP-MS
According to previous reports, 21 the relationship between the number of total atoms in each nanoparticle can be calculated as Formula a where N represents the number of atoms in each nanoparticle, while is the metal density, is atomic weight and represents the diameter. In sp-ICP-MS, the number of atoms is proportional to intensity ( ) which is illustrated in Formula b, thus the relationship between diameter ( ) and intensity can be calculated as Formula c ( is a constant) where intensity is proportional to .

Fig. S8: Labeling of AuNPs and PtNPs
DLS was applied for characterization of nanoparticles labeling. In accordance with the results, distribution of nanoparticles moved to bigger diameters, indicating that antibodies were loaded on the surface of nanoparticles. In addition, no changes in intensity of nanoparticles after labeling in sp-ICP-MS measurements, further proving the successful labeling.

Fig. S9: Optimization on Volume of Magnetic Beads in Immunoassay
Optimization on volume of magnetic beads was carried using fPSA-PtNPs immunoassay. As shown below, both Pt and Fe signals increased when using bigger volume of magnetic beads. By evaluating the signal-to-noise ratios, the highest S/N signals were found using 5 µL magnetic beads. Lower volume of magnetic beads was also considered in optimizations but the separation in sample became difficult in return. Thus, we finally chose 5 µL magnetic beads in each sample (about 5 µg magnetic beads per sample).

Fig. S10: Optimization on Wash Times of Magnetic Beads in Immunoassay
Optimization on wash times of magnetic beads was also carried using fPSA-PtNPs immunoassay. According to the results, S/N signals reached the maxima when taking two times wash steps.

Fig. S13: Frequency distribution of Pt/Au content on Single Magnetic Bead
After measuring magnetic beads by sp-ICP-MS under the different concentrations of relative antigens, frequency distributions were processed by applying the intensity of Au or Pt per magnetic bead as X coordinate, and the number of magnetic beads as Y coordinate. The background signals were subtracted to get the metal distributions. On the obtaining histograms, the first sample distribution (0 ng/mL) attributed to the signals of background and a small number of nanoparticles left in sample after two-steps washing.

Fig. S14: Selectivity of the SMB Nanoplatform
Selectivity of the proposed SMB nanoplatform was carried by mixing nanoparticle probes and magnetic beads with tPSA/fPSA or interferences. Detail information was illustrated in Experimental Procedures part.

Fig. S15: ROC Curves for Prostate Disease Differentiation
Predictive prognostic values of fPSA (Green), tPSA (Red) and Pt/Au Ratio (Blue) using ROC curves in differentiation of healthy, BPH and cancer groups.