Supporting Online Materials for
Ultra-Sensitive Immunoassay for Prostate Specific Antigen
Using STM Based Electrical Detection
Jeong-Woo Choi, Byung-Keun Oh, Yong-Hark Jang, Da-Yeon Kang
* To whom correspondence should be addressed. E-mail : jwchoi@sogang.ac.kr
SUPPORTING ONLINE MATERIALS
Preparation of antibody fragment
Monoclonal (Mab) and polyclonal antibody (Pab) fragment against PSA was utilized for the
immobilization on Au surface. The preparation method of antibody fragment was illustrated
on the basis of chemical reduction using 2-mercaptoethylamine (MEA). The method is
similar to that presented in the reference.1 MEA was applied to antibody solution for the
fragmentation of immunoglobulin G (IgG) molecules. After incubation for 90 minutes under
37 oC, the residual MEA was removed through the dialysis membrane with molecular cut-off
membrane against phosphate buffered saline (PBS) - ethylenediaminetetraacetic acid (EDTA)
buffer (pH 7.4) which is PBS containing, 5 mM of EDTA. Fig.S1 shows the result by SDSPAGE
Fig S1.
SDS-PAGE
Fabrication of Au nanoparticle-antibody conjugates
For the fabrication of Au nanoparticle-antibody conjugate, coagulation test was carried out
ahead in order to find out the optimal concentration of antibody solution which minimizes the
disturbance of the nanoparticle-antibody complex. In this study, mixed solution of Au
nanoparticle and antibody fragment was prepared after dilution with water, of which the
volume ratio of water, Pab against PSA (0.1 mg/mL), and Au nanoparticle was 6:3:1 based
on 10 mL volume. The prepared solution was incubated for 2 hours at 4oC. For the
stabilization of Au nanoparticle-antibody conjugate, 0.1 ml of 5% casein was added in the
mixed solution and it was incubated for 1 hour at 4oC. The prepared Au nanoparticleantibody conjugated was centrifuged at 34,000 rpm for 1 hour at 4oC in Beckman
ultracentrifuge (Optima XL-90, Beckman Instruments, Inc., CA, USA) equipped with a
SW50.1 swing bucket rotor. After centrifugation, the sediments corresponding to Au
nanoparticle-antibody complexes were recovered and resuspended in 0.5 mL of PBS. The
detection of nanoparticle-antibody conjugate was using UV/vis spectrophotometry. The
nanoparticle has an intrinsic property that absorbs the light at the visible wavelength. Au
nanoparticle absorption wavelength was observed to be 525nm. And then protein such as
antibody has an absorbance at the wavelength of 280nm due to peptide bonding (Fig S2).
Fig S2. Confirmation of Au nanoparticle-antibody conjugate by UV-vis. spectroscopy
Scanning tunneling microscopy (STM)
The surface topography of the prepared biosurface was obtained by commercially available
scanning tunneling microscopy (STM, XE-100, Park Systems, Korea). And the substrate was
fixed using silver phase on the sample disk for current flow among sample, sample disk and
tip of STM. Information with respect to surface topography and current profile was acquired
under the condition of scan size = 500nm2, scan rate=0.3Hz, Iset = 0.5 nA, and Vbias = 0.5 V
when the STM investigation was carried on the surface with Au nanoparticle-antibody
conjugates. The resolution of all topography and current profile was same as 256pxl ×256pxl.
Power spectra of the current profiles acquired in STM images
The basic principle of scanning tunneling microscopy is to scan a lot of discrete points with a
measurement of tunneling current between tip and conductive surface. This scanning
generates a set of tunneling currents for the display of topography, which is virtually a
function of time with constant interval. In practice, a massive amount of data is obtained in
order to convert raw current signals into topography. When the tip is scanning over the
surface with Au-nanoparticle, these electrical signals contain a lot of pulse-like peaks due to
the difference of I-V property. The obtained current signals can be simplified by means of the
analysis in frequency domain. Because the set of current signals is a function of time, we
were able to do Fourier-transformation in order to investigate its characteristics in frequency
domain.
Based on the random distribution of Au-nanoparticle on surface, the frequency of generated
pulse-like peaks is proportional to the surface density of Au-nanoparticle. It also allows the
peak-to-peak distance to be shortened, which will reflect the higher signal intensity in short
period region. Periodogram (an estimate of a spectral intensity of signal) could give us (semi) quantitative information about the density of localized Au nanoparticle on surface. It can be
directly extracted from Fourier-transformed data through simple mathematic manipulation.
The overall analysis process is consistent with motion of biochemical binding event in nanoscale domain which were already published.2 In order to clarify the variation trend of
periodogram, non-linear regression curve which would best fit was obtained with the discrete
data points. MicrosoftTM Excel software was utilized for the non-linear regression. The
manipulated regression curves were displayed in Fig S3(b).
Tunneling current profile of dispersed Au nanoparticle on the surface
For the proof-of-principle of the suggested electrical detection, a simple model system was
investigated with Au nanoparticle and self-assembled dithiolated organic molecule (1,8octanedithiol, ODT). Au nanoparticle with 5 nm of diameter was immobilized on the ODT
monolayer. Localized binding event of Au nanoparticle was observed as shown in Fig. S3(a).
The surface density of the Au nanoparticle immobilized on ODT self-assembled monolayer
(SAM) was proportional to the added concentration of Au nanoparticle. In case that
concentration is over a critical limit, it can ultimately form a thin film.3 The current profile
obtained for the display of STM topography was utilized as input variables for periodogram
analysis, which is an approximation of power spectrum. Fig. S3(b) shows the periodograms
with respect to the bare Au surface and Au nanoparticle-immobilized surface. When lots of
Au nanoparticle were immobilized on surface (0.01 wt %), the reduced interval between
nanoparticles would result in high frequency of the current peaks. It causes the dimensionless
intensity in periodogram to be significantly increased at the short period region. Obviously
the regression curve of the periodogram corresponding to 0.01 wt% of Au nanoparticle could
be distinguished with that of bare Au. The magnitude of dimensionless intensities was
changed according to the applied concentration of Au nanoparticle. At a distinct point, less
than /4 period, we could correlate this dimensionless intensity to the concentration of the Au
nanoparticle. At this point of /4 period, the relationship between the Au nanoparticle
concentration (notice that the x-axis is a log scale) and the dimensionless intensity was linear
over a concentration range of four orders of magnitude (Fig. S3(b), inset). Therefore, the
suggested detection technique shows that semi-quantitative measurement for the surface
density of Au nanoparticle can be done by means of periodogram analysis. The error and the
deviation of inset of Fig. S3(b) come from the arbitrary decision of data collection point. The
periodogram within /4 shows more significant separation which enables us to do more
sensitive detection. Because the peak-to-peak distance is getting shorter as the surface density
of Au-nanoparticle increases, much higher signal intensity is shown at the close to the zero
point. The random distribution of Au-nanoparticles may also result in the error and the
deviation. Each data set has a unique ensemble with locally immobilized Au-nanoparticle.
Even though we had a lot of experiments in order to obtain reliable data for quantitative
measurement, the error and deviation would be inevitable which would be intrinsic property
of the proposed detection technique. And probably, the surface roughness of gold surface
plays a role in making noise in signal.
Fig S3. (a) Current profile of Au nanoparticle layer immobilized on ODT SAM with
different particle concentration (b) Power spectra of the current profiles acquired in the
corresponding STM images (insert: calibration curve from the power spectra of the
current profiles)
Comparison to electrical signal of prostate specific antigen (PSA) and Human serum
albumin (HSA)
In order to investigate non-specific binding event of the biosurface functionalized with antiPSA fragment to HSA, HSA was applied to the biosurface with anti-PSA fragments. The
comparison to electrical signal to PSA and HSA was shown in the Fig S4. Although the
concentration is same, intensity of the power spectrum was remarkably different. From these
experimental results, we could confirm the specific binding of anti-PSA functionalized
biosurface to PSA.
Fig S4.comparision to signal of PSA and HSA
References
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(2000)
2. S. Kossek, C. Padeste, L. X. Tiefenauer, H. Siegenthaler, Biosens. Bioelectron. 13, 31
(1998)
3. Y. Jin, X. Kang, Y. Song, B. Zhang, G. Cheng, S. Dong, Anal. Chem. 73, 2843 (2001)