Two different sequences of adenosine aptamers, the 27-mer and 35-mer (sequences listed in Table 1 ) were used as the aptasensor receptors. All aptamers were functionalised onto the CNT side walls using 1-pyrenebutanoic acid succinimidyl ester (PBASE) as a molecular linker [34 (link)]. The fabricated CNT FETs were submerged into 1 mM PBASE (95%, Sigma Aldrich, St. Louis, MO, USA) in methanol solution for 1 h, then rinsed a few times in pure methanol to remove excess PBASE and washed in DI water for 5 s. Meanwhile, the aptamers were diluted to a concentration of 1 µM aptamer solution in 20 mM Tris-HCl buffer (Sigma Aldrich, St. Louis, MO, USA) and then denatured at 70 °C for 5 min in an oven. A total of 100 µL of the prepared aptamer solution was added onto the channel of the CNT FET at room temperature overnight in a closed petri dish. Finally, the unbound aptamers on the surface were removed by washing the devices with 20 mM Tris buffer and DI water before drying in N2.
Pbase
Manufactured by Merck Group
Sourced in United States
PBASE is a laboratory equipment product developed by Merck Group. It is a precision instrument designed for accurate and efficient sample preparation and analysis. The core function of PBASE is to provide a reliable and consistent platform for various laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
N n dimethylformamide (dmf), by Thermo Fisher Scientific (2 mentions)
Anti cd63 antibody, by BD (1 mentions)
N n dimethylformamide (dmf), by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Pbase, by Thermo Fisher Scientific (1 mentions)
Tris hcl buffer, by Merck Group (1 mentions)
Mucin, by Merck Group (1 mentions)
Nahco3, by Merck Group (1 mentions)
Sodium chloride (nacl), by Merck Group (1 mentions)
Na2hpo4, by Merck Group (1 mentions)
Potassium chloride (kcl), by Merck Group (1 mentions)
Cacl2, by Merck Group (1 mentions)
Nah2po4, by Merck Group (1 mentions)
10 protocols using pbase
1
Aptamer-Functionalized CNT FET Biosensor
2
Graphene Sensor Functionalization for SARS-CoV-2 Antibody Detection
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Graphene sensors (#GPH381-2, PreDiagnose, Karlslunde, Denmark) was functionalized by pipetting 50 µL 2 mM 1-Pyrenebutyric acid N-hydroxysuccinimide ester (PBASE) (#114932-60-4, Sigma-Aldrich, St. Louis, MO, USA) dissolved in 1:3 v/v of dimethylformamide (68-12-2, Sigma-Aldrich) to methanol (#67-56-1, Sigma-Aldrich) onto the sensor to cover the working electrode. Sensors were covered to avoid evaporation and incubated for 2 h at room temperature. The sensors were then washed with methanol and dried with nitrogen. The anti-spike antibody (#45150-D003, Sino biological) was diluted 100x in PBS to approximately 22 µg/mL, and 10 µL was added to the working electrode and incubated for 2 h at 37 °C and 300 rpm shaking. After washing and drying the sensors with PBS, they were blocked by the addition of 50 µL PBST (0.05% Tween-20) containing 1% BSA to the working electrode, and sensors were incubated for 1 h at room temperature. The sensors were then washed with deionized water and then rinsed by 5 s immersion in 37 °C PBST on a magnet stirrer at 400 rpm followed by drying with nitrogen. After the blocking step, the electrochemical signal of the functionalized sensor was measured using a potentiostat.
3
DNA Probe Immobilization on Graphene FET Array
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The GFET array was soaked
in a 1 mM PBASE (Sigma-Aldrich) in DMF (Fisher) solution for 20 h
and then washed thoroughly with DMF, IPA, and DI water, each for 3
min. The array was then incubated in a 1 μM aqueous solution
of 22-mer probe-DNA (5′-amine-CCCAACAACATGAAACTACCTA-3′),
40-mer probe-DNA (5′-amine-AATTACAAAAACAAATTACAAAAATTCAAAATTTTCGGGT-3′),
or 60-mer probe-DNA (5′-amine-AAACTAAAGAATTACAAAAACAAATTACAAAAATTCAAAATTTTCGGGTTTATTACAGGG-3′)
for 3 h and rinsed with DI water. Following probe attachment, an aqueous
solution of target or control DNA with known concentration was pipetted
onto the chip, and incubated in a warm, humid environment for 30 min
to suppress evaporation. The chip was rinsed with DI water and dried
with compressed nitrogen before performing electrical measurements.
in a 1 mM PBASE (Sigma-Aldrich) in DMF (Fisher) solution for 20 h
and then washed thoroughly with DMF, IPA, and DI water, each for 3
min. The array was then incubated in a 1 μM aqueous solution
of 22-mer probe-DNA (5′-amine-CCCAACAACATGAAACTACCTA-3′),
40-mer probe-DNA (5′-amine-AATTACAAAAACAAATTACAAAAATTCAAAATTTTCGGGT-3′),
or 60-mer probe-DNA (5′-amine-AAACTAAAGAATTACAAAAACAAATTACAAAAATTCAAAATTTTCGGGTTTATTACAGGG-3′)
for 3 h and rinsed with DI water. Following probe attachment, an aqueous
solution of target or control DNA with known concentration was pipetted
onto the chip, and incubated in a warm, humid environment for 30 min
to suppress evaporation. The chip was rinsed with DI water and dried
with compressed nitrogen before performing electrical measurements.
4
Graphene FET Surface Functionalization
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Samples were first incubated
for 2 h with 3 mM PBASE (Sigma-Aldrich) in dimethylformamide (DMF)
(Sigma-Aldrich) at room temperature. Then DMF and DI water were used
to gently rinse the GFET sample to remove excessive PBASE from the
surface and dried with N2. Then, the anti-CD63 antibody
(BD Bioscience US) were used. The antibodies were supplied in a stock
solution of 0.5 mg/mL in an aqueous buffer solution (containing ≤0.09%
sodium azide) and were diluted using 1 × PBS to a concentration
of 100 μg/mL. Droplets of 20 μL of 100 μg/mL anti-CD63
antibody were placed on the surface and left overnight in a humidified
environment at 4 °C. The samples were then sequentially rinsed
with 1 × PBS and DI water, and dried with N2.
for 2 h with 3 mM PBASE (Sigma-Aldrich) in dimethylformamide (DMF)
(Sigma-Aldrich) at room temperature. Then DMF and DI water were used
to gently rinse the GFET sample to remove excessive PBASE from the
surface and dried with N2. Then, the anti-CD63 antibody
(BD Bioscience US) were used. The antibodies were supplied in a stock
solution of 0.5 mg/mL in an aqueous buffer solution (containing ≤0.09%
sodium azide) and were diluted using 1 × PBS to a concentration
of 100 μg/mL. Droplets of 20 μL of 100 μg/mL anti-CD63
antibody were placed on the surface and left overnight in a humidified
environment at 4 °C. The samples were then sequentially rinsed
with 1 × PBS and DI water, and dried with N2.
5
Quantitative Detection of Tumor Markers
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The monoclonal anti-alpha-fetoprotein (anti-AFP), AFP, hCG, and CEA were purchased from Antibody Center (Seongnam, Korea). The human plasmas of HCC patients were provided at Keimyung University school of Medicine (Daegu, Korea). The concentration of AFP in each HCC patient plasma was verified at Keimyung University school of Medicine and HCC patient plasma were used without any purification process. PBASE and bovine serum albumin (BSA) were purchased from Sigma-Aldrich (Seoul, Korea). Large-sized graphene on a PET substrate was purchased from MCK Tech (Ansan, Korea). Ultrapure water (18.2 MΩ·cm) was used for the preparation of all solutions. PBS was made in the laboratory and was prepared using 137 mM NaCl, 8.1 mM Na2HPO4·12H2O, 2.7 mM KCl, and 1.5 mM KH2PO4. 0.01 × PBS (pH 7.4) was prepared by diluting 1 × PBS with ultrapure water. Antigens and antibodies were diluted in 1 × PBS.
6
Immobilization of Variant Receptor on GFETs
7
Functionalization of Graphene-based Biosensor
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The functionalization process was carried out as follows. To immobilize the probe DNA to graphene surface without introducing defects, PBASE was used as a linker47 . Next, 10 mM PBASE solution in N,N-dimethylformamide was introduced into sensing channels through the inlet tube and to soak graphene for 1 h at room temperature, after which the sensing channels were rinsed three times by the pure N,N-dimethylformamide methanol to wash away any excess reagents. For the immobilization of probe DNAs, single-stranded 1-μM 5′-amine-modified DNA in 1 × PBS buffer flowed through the sensing channel for 1 h. Nonbound DNAs were washed thoroughly with 1 × PBS buffer. Then, 100 mM ethanolamine solution was released into the sensing channel through the inlet tube to deactivate and block the excess reactive groups remaining on the graphene surface. Here 1 × PBS is the standard PBS solution and 0.1 × PBS, 0.05 × PBS, 0.01 × PBS and 0.005 × PBS are diluted from 1 × PBS by ultrapure water. DNA samples were purchased from Sangon Biotech. PBASE and ethanolamine were purchased from Sigma Aldrich. All salts were purchased from Sigma (Sigma Ultra grade) and dissolved in ultrapure water.
8
Graphene Channel Functionalization with IgG Aptamer
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The graphene channel was then functionalized with 5’-amino modified human immunoglobulin G (IgG) aptamer using 1-pyrenebutanoic acid N-succinimidyl ester (PBASE) (Sigma-Aldrich) as the linker. Briefly, PBASE (10 mM, dissolved in dimethyl sulfoxide (DMSO)) was injected into the channel, and kept for 2 h. After rinsing with DMSO, 5′-amino modified IgG aptamer (100 μM in 1× PBS (10 mM); Base Pair Biotechnologies, Inc., Pearland, TX, USA) was injected into the channel and incubated for 3 h, to allow the conjugation with PBASE. The remaining unconjugated sites were then blocked by bovine serum albumin (BSA, 10% w/v in 1× PBS). After functionalization, the channel was rinsed with 1× PBS and kept in 1× PBS in refrigerator until usage.
9
Graphene Aptamer Biosensor Functionalization
10
Purification and Binding of SARS-CoV-2 Spike
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GFET-S20 array and CVD-G on Si/SiO 2 were produced and provided from Graphenea Semiconductor SLU. PBASE, ACE2, mucin, Na 2 HPO 4 , NaH 2 PO 4 , NaHCO 3 , KCl, NaCl, CaCl 2 and EtOH were purchased from Sigma Aldrich. Ecto S of SARS-CoV-2 was purchased from CIC bioGUNE. PBASE and ACE2 were stored at -20 °C, while Ecto S at 80 °C. The Ecto S (BEI construct NR-52394) was expressed by transient transfection of HEK293F suspension cells and purified from clarified cell supernatants seven days post-transfection using a nickel affinity column and size-exclusion chromatography as previously described. 32 Then, the Ecto S and previously purified ligand according to Barnes et al., 2020 were mixed and incubated 2 hours at 4 °C.
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