Materials
11-Mercaptoundecanoic acid (MUA), 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), N-hydroxysuccinimide (NHS), bovine serum albumin (BSA), human serum, streptavidin–alkaline phosphatase (STA-AP), streptavidin–horseradish peroxidase (STA-HRP), nitro blue tetrazolium (NBT), and 5-bromo-4-chloro-3-indolyl phosphate p-toluidine (BCIP) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Skim milk was purchased from BD Difco (Franklin Lakes, NJ, USA). UV-curable perfluoropolyether (PFPE), which is used for replication of the master and imprint resin, and mr-I PMMA 35k200 were purchased from Solvay Solexis (Marshalton, DE, USA) and Micro Resist Technology (Berlin, Germany), respectively. Biotin XX Tyramide SuperBoost kit was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Recombinant SCG2 (Cat# MBS2028995), biotinylated anti-SCG2 antibody (Cat# MBS7046117) were obtained from MyBioSource (San Diego, USA). Scg2 capture antibody was manufactured as below: anti-SCG2 polyclonal antibody was raised against the peptide AHKEESSPDYNPYQG (human SCG2, aa 69–83) as an antigen in rabbit and purified from rabbit serum using antigen peptide covalently linked with Sulfolink coupling gel (Thermo Fisher Scientific). Other antibodies were purchased from commercial sources: rabbit anti-SCG2 (Cat# ab126935, Abcam, Cambridge, UK), mouse anti-PSD-95, rabbit anti-vGlut1 (Cat# MA1-045, Thermo Fisher Scientific), mouse anti-gephyrin (Cat# 147 111, SYSY, Goettingen, Germany), rabbit anti-vGAT (Cat# 131 002, SYSY). The Cy3, FITC, and HRP-conjugated secondary antibodies were obtained from Jackson ImmunoResearch (West Glove, PA, USA).
Clinical human serum samples
An information sheet describing the rationale of the study and individual rights was handed to the parents of the participants. Written informed consents were then obtained for the blood samples and all experiments were performed in accordance with the approved guidelines and regulations of Human Ethics Committee and the Institutional Review Board (IRB) of Samsung Medical Center (IRB number: 01–201,604-31–003). All patients were diagnosed with global developmental delay according the guidelines of Diagnostic and Statistical Manual of Mental Disorders: Fourth Edition-V (DSM-V)1. The whole blood collected from participants was allowed to clot at room temperature (RT), and then the clot was removed by centrifuging at 2,000 × g for 10 min in a refrigerated centrifuge.
SCG2 detection using conventional ELISA
The conventional ELISA method was used as the standard in this study. Typically, 100 µL of the homemade anti SCG2 antibody (2 μg/mL) was coated in 96-well plates overnight at 4 °C, and then the wells were blocked with skim milk (w/v, 5%) for 2 h at RT. After blocking, the wells were washed with PBST (PBS at pH 7.4 containing 0.1% Tween20) to remove excess skim milk. Next, 50 μL of various concentrations of SCG2 in human serum were added to the wells. The patient serums were diluted fivefold with PBS buffer. The wells were incubated at RT for 2 h and washed thrice. Later, 100 μL of biotinylated anti-SCG2 antibody solution was added to each well and incubated at RT for 1 h. The wells were washed again, and 100 μL of STA-HRP (500 ng/mL) was added to the wells and incubated at RT for 30 min. Then, the wells were washed with PBST four times, and 100 μL of TMB substrates were introduced and incubated at RT for 30 min. Finally, 50 μL of stop solution (2 M H2SO4) was added, and the optical density was measured at 450 nm using a microplate reader.
Sensor chip fabrication
The sensor chip fabrication procedure was reported in our previously works21. Briefly, in order to make a sensor chip, the silicon master with the nano-hole pattern used as the master mold had a diameter, pitch, and depth of 150 nm, 300 nm, and 150 nm, respectively. The master surface was covered with a polycarbonate (PC) film after the UV resin (PFPE) to be used for the replica mold was dropped onto the surface. The PC film of the master was pressed with a hand roller to fill the hole pattern with resin, and then the replica mold was cured on the PC film. A thermoplastic resin (PMMA) was spin-coated on a glass substrate for 2 h at 130 °C (5 bar). The replica mold was separated from the substrate after cooling to 90 °C. To protect the top region of the imprinted resin from O2 plasma etching, a 30 nm layer of titanium was deposited at a tilted angle of 70° during rotation in an e-beam evaporator. The residual layer was removed by O2 plasma etching (PINK GmbH Plasma-finish, Bestenheid, Germany) at an O2 flow rate of 300 mL/min and power of 300 W for 6 min. Finally, a Ti/Au layer (1/20 nm) was deposited by e-beam evaporation, and lift-off was carried out using an acetone solution for 5 min. There are four regions with a diameter of 3 mm in one chip; one of the four regions was used as the reference channel, and the remaining three regions were used as the sample channels.
SCG2 detection by signal enhanced nanoplasmonic immunosensor
The fabricated gold nanodot array (GNA) chips were cleaned with piranha solution (H2SO4/H2O2 = 3:1) at 90 °C to eliminate the impurities on the surface of the chips. The chips were washed with deionized water (DW) and dried with nitrogen gas. The cleaned chips were immersed in a 10 mM 11-mercaptoundecanoic acid (MUA) solution for 12 h. The carboxylate groups of the MUA surfaces were activated in a solution of 0.1 M 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and 0.025 M of N-hydroxysuccinimide in DW for 9 min. Then, 0.1 mg/mL anti-SCG2 capture antibody in PBS was added to the GNA chip and incubated at 4 °C overnight. After washing the chip 3 times with 50 μL PBST, blocking solution (5% skim milk) was applied for blocking nonspecific binding, followed by incubation at 37 °C for 30 min. 50 μL of recombinant SCG2 at various concentrations (0–40 ng/mL) in human serum was injected, followed by incubation at 37 °C for 1 h. After incubation, the chips were washed with 3 times with PBST. For nanoplasmonic immunosensor, 5 μg/mL of biotinylated anti-SCG2 antibody and 5 μg/mL STA-AP were added to the chip. After the incubation for 1 h, the excess detection antibody and STA-AP were removed by washing the chips 3 times with washing buffer. Next, the enzyme-catalyzed precipitation reaction was triggered by adding 0.1 M 5-bromo-4-chloro-3-indolyl phosphate p-toluidine (BCIP) and 1 mg/mL nitro blue tetrazolium (NBT) in an AP buffer (100 mM Tris–HCl, 100 mM NaCl, 5 mM MgCl2) at pH 9.5 for 30 min. For enhanced nanoplasmonic immunosensor, 5 μg/mL of biotinylated anti-SCG2 antibody and 5 μg/mL of STA-HRP instead of STA-AP were added to the GNA chip. The chip was washed, and then 1 × biotin-tyramine and 0.03% H2O2 in 0.1 M borate buffer (pH 8.5) were added to the chip followed by keeping at room temperature for 15 min. After washing, 5 μg/mL of STA-AP was added to the chip and incubated at 25 °C for 30 min. Unbound STA-AP was removed by washing the chips 3 times with PBST. The enzyme-catalyzed precipitation reaction was triggered by adding 0.1 M BCIP and 1 mg/mL NBT in an AP buffer at pH 9.5 for 30 min. LSPR wavelength change was measured by LSPR observation setup with the back-reflection mode and determined by the centroid fit using a homemade program based on LabVIEW21. The scan area was the 1 mm diameter part located in the center of each region, and it was passed through the light source as described previously21.
DNA constructs
For knockdown of rat SCG2 (NM_022669.2), nt 530–547 (GGTTCCCTCTCATGTATG, SCG2-shRNA) was subcloned into pSuper.gfp/neo (OligoEngine, Seattle, WA, USA). Full-length human SCG2 (NM_003469, aa 1–617) was subcloned into GW1-CMV, and used as Rescue SCG2 for SCG2-shRNA.
Immunoblot analysis
Immunoblotting was performed as previously described42. This study was performed in accordance with the regulations outlined by the Korean law. The animal experiment protocols were approved by the Animal Use and Care Committee of Korea Research Institute of Bioscience and Biotechnology (Permit Number: KRIBB-AEC-19115). Animals were sacrificed using CO2 gas, and all efforts were made to minimize suffering. Briefly, mice were sacrificed and the brain tissue was quickly removed and homogenized in a homogenization buffer (50 mM Tris–HCl, 150 mM NaCl, 1% Nonidet P-40, 0.1% SDS, and 0.1% sodium deoxycholate, pH 8.0) containing protease inhibitor cocktail (Roche, Mannheim, Germany). Protein samples were resolved with SDS-PAGE and then transferred onto a polyvinylidene fluoride membrane (BioRad Laboratories, CA, USA). Blots were incubated with primary and secondary antibodies followed by visualization using an Enhanced Chemiluminescence kit (Atto Corp., Japan). Immunoblot images were quantified using Quantity One 1-D analysis version 4.6.1 software (Bio-Rad Laboratories) or Image J software (NIH) (https://imagej.nih.gov/ij/index.html).
Transfection of neurons and immunocytochemistry
Primary hippocampal neurons were prepared from embryonic day 18 rats as described previously29. Briefly, hippocampi were dissected with trypsin and plated on coverslip coated with poly-L-lysine in Neurobasal medium (Thermo Fisher Scientific) supplemented with B27 (Thermo Fisher Scientific). After 2–3 h of incubation, the plating medium was changed with a growth medium (plating medium and glutamate). Cultured hippocampal neurons were transfected by the calcium phosphate method at 5 or 7 days in vitro. For immunofluorescent staining, after 7–9 days of transfection, cultured hippocampal neurons were fixed in 4% (v/v) formaldehyde/ 4% (w/v) sucrose, and permeabilized with 0.2% (v/v) Triton X-100 in phosphate-buffered saline followed by incubation with primary antibodies and fluorophore-conjugated secondary antibodies.
Image analysis and quantification
Images captured by confocal microscopy (LSM 810, Zeiss, Gottingen, Germany) were analyzed blindly using MetaMorph software (Version: 7.10.1.161, Molecular Devices, San Jose, CA, USA). Sholl analysis of dendritic arbors was performed employing the modified method of Nakayama et al.43. After images of individual neurons were printed, printouts were placed under a clear sheet featuring concentric circles with diameters increasing in 20 µm increments. The center of the circles was placed at the cell body center and the numbers of dendrites crossing each concentric circle were counted. Quantification of primary and secondary dendrites was also performed by imaging of individual neurons. The density of dendritic spine and synaptic protein clusters were measured from 27 to 32 dendrites of 7 ~ 9 neurons.
Statistical analysis
GraphPad Prism software (GraphPad Software, Inc., La Jolla, CA, USA) was used to perform all statistical analyses. Two-sample comparisons were conducted with Student’s t-tests, while multiple comparisons were performed with a one-way analysis of variance (ANOVA) followed by Tukey post hoc tests. All results are presented as the means ± s.d. or means ± s.e.m. Differences with a p value less than 0.05 were considered to be statistically significant.
