Mol. Cells 2016; 39(11): 807-813
Published online November 30, 2016
https://doi.org/10.14348/molcells.2016.0167
© The Korean Society for Molecular and Cellular Biology
Correspondence to : *Correspondence: dklee@skku.edu (DKL); skim@dongguk.edu (SK)
Keywords aptamer, biolayer Interferometry, biosensor, cell-SELEX,
Food and water contamination by pathogenic bacteria is a serious threat to the human health. Therefore, fast, accurate and economic methods of detection are critical to the timely diagnosis of virulent strains in the infected sources (Koedrith et al., 2015). As many pathogens are not easily detected, indicator organisms are a fundamental monitoring tool to assess the microbiological status of water and food (Tortorello, 2003). Detection of safety indicators suggest the presence of conditions associated with increased risk of exposure to a pathogen. These, along with yeasts, molds and some other bacterial species, also include
Conventional methods of
Unless otherwise noted, all chemicals were purchased from Sigma Aldrich or invitrogen. SELEX library (N40 library) was obtained from IDT and transcribed using DuraScribe® T7 Transcription Kit (Epicentre Biotechnologies, USA) following the manufacturer’s protocol. Truncated aptamer Ec3(31), biotinylated or thiol modified oligonucleotides, HSR-1, SQ2 Mutant were chemically synthesized by ST pharma. Inc. Oligonucleotide sequence details are provided in Supplementary table 1. Lipoteichoic Acid (LTA) aptamer (OCT biotech, USA) was used as a positive control for gram positive bacteria and Lipopolysaccharide (LPS) aptamer (OCT biotech, USA) and
The starting 2′-F pyrimidine modified RNA library was prepared by
The screening of potential aptamers and calculation of binding affinity was done using qRT-PCR. The four selected aptamer sequences were
Truncated aptamer Ec3(31) with 3′ biotin, SQ2 mutant-biotin and N40 primer-biotin controls, were allowed to bind to 20 μl of streptavidin coated magnetic beads (dynabead, Invitrogen, USA) for 15 min at RT. yeast tRNA (0.1 mg/ml) and BSA (1 mg/ml) were added and the incubation was further continued for 45 min. Unbound aptamer were washed-off and this preblocked bead-aptamer complex were incubated with various cell density of
ForteBio’s Octet RED 384TM instrument and streptavidin (SA) sensors (Pall life sciences, USA) (Cat: 18-5019) were used to study interactions between aptamer candidates and bacteria cells or bacteria lysates, according to manufacturer’s instructions. All the aptamer target interactions were performed in binding buffer. In the case of antibody and control aptamers (LTA and LPS), 1X kinetic buffer (Fortebio) and buffer recipe provided by OTC biotech were used in accordance with the manufacturer’s protocol. All aptamer candidates were heated at 95°C, chilled on ice for 5 min, and suspended in binding buffer at RT prior to use. SA sensors were pre-wet by binding buffer for 30 min followed by loading with aptamer candidates at 25°C for 1200 s at 1000 rpm. After washing out nonspecific binding aptamers, sensors were blocked with biocytin and dipped into bacteria cells and lysates with desired concentrations. Association and dissociation profiles were measured for 10 min. To monitor non-specific binding for biosensor, a reference sensor (without aptamer loading) was performed with the same procedure as aptamer loaded sensors. Kinetic screening results were acquired by subtraction from the reference sensor using Octet data analysis software, version 7.0 (ForteBio). In the case of aptamer inhibition, SA sensors were pre-coated with biotinylated truncated aptamers and then associated with lysates and unlabeled aptamer mixtures (pre-incubation at RT for 20 min before BLI assay).
Two Cr/Au Gap capacitance electrodes of thickness 5 nm and 15 nm respectively were deposited on Pyrex glass (7740) by thermal evaporation process. The Cr/Au layer were patterned and etched as shown in Fig. 4A. Parylene was deposited at 2 μm thickness at each electrode and patterned to form flow channels. One electrode makes inlet and outlet holes for sample introduction and removal, and two parylene coated electrodes are finally bonded by thermo-compressing bonding (150°C, at 24 MPa for 20 min).
Thiol modified and Cy3 labeled Ec3 aptamer were mixed with 10 mM NaCl. Four concentrations of aptamer solution were tested for immobilization (40 pM, 0.4 nM, 4 nM and 18 nM) and incubated for 1 hour. The intensities of fluorescent signal, representing the aptamer immobilization capacity, were monitored. The unbound sample was washed by injection of PBST (0.1% tween 20 in PBS buffer). The chip was heated to 90°C and slowly cooled down to allow proper aptamer secondary structure formation. The chip was washed with DI water thoroughly to remove the unbound aptamer. 1 mM BSA solution was introduced into the channel and incubated for 30 min. Serial dilutions of bacteria solution (108, 106, and 104 CFU/ml) was introduced onto the chip. After 1 h of incubation, the chip was washed thoroughly and the bound bacteria were stained with Sybr Gold and visualized under fluorescent microscopy (Nikon eclipse TE2000-U). For impedance analysis, EIS measurement were performed which swept frequency from 1 Hz to 1 MHz under low applied voltage (less than 0.01 V) to avoid electrochemical reaction between electrode and liquid solution. As a negative control, we also measured impedance of buffer solution as a function of frequency.
12 rounds of selections were performed to obtain
In order to check the potential of aptamer Ec3 as an
Biolayer interferometry or BLI is an optical analytical technique that allows real-time monitoring of molecular binding events occurring on the surface of the biosensor (Concepcion et al., 2009). BLI has been shown to be an efficient label-free assay format to detect molecular interactions. (Kammer et al., 2014; Zichel et al., 2012). It analyzes the pattern of white light reflected from two surfaces: a layer of immobilized protein on the biosensor tip, and an internal reference layer. Any change in the number of molecules bound to the biosensor tip causes a shift in the interference pattern that can be measured in real-time. Use of BLI in detection of whole pathogens is a very novel approach and not many studies have been done in this regard. In a recent and probably the first study where BLI has been used to detect whole bacteria, gram-negative bacterium
To further improve upon the sensitivity of aptamer mediated
Nyquist plot from impedance spectroscopy of various concentrations of bacteria solution and negative control are given in Fig. 4C. Each impedance signal is clearly different to that of negative control (No bacteria). Impedance increases with bacterial concentration. (Supplementary Fig. 4). In low frequency range such as 1 Hz, impedance increase is caused by capacitance, since phase angle is close to - 90 deg. However, at higher frequency (over 1 kHz) resistance increases in proportion to the number of bacteria in solution. When frequency is over 10 kHz, impedance value is close to constant (Supplementary Fig. 4) and it increases in accordance with the density of bacteria concentration. It suggests that bacteria binding onto the electrode work as resistance particles, inhibiting current flow between electrodes. The system that we developed here can be used as a sensor to monitor
The 2′F modified RNA aptamer developed in this study has far better
Although our assay sensitivity is lesser in comparison to some of the impedance based detection of
Aptamer sequences identified
Sequence family | Aptamer sequence | % of total sequences |
---|---|---|
Ec3 | AUACCAGCUUAUUCAAUU | 20 |
Ec2 | AUACCAGCUUAUUCAAUU | 7 |
Ec5 | AUACCAGCUUAUUCAAUU | 6.7 |
Mol. Cells 2016; 39(11): 807-813
Published online November 30, 2016 https://doi.org/10.14348/molcells.2016.0167
Copyright © The Korean Society for Molecular and Cellular Biology.
Pooja Dua1,4, Shuo Ren2,4, Sang Wook Lee2,4, Joon-Ki Kim1, Hye-su Shin1, OK-Chan Jeong3, Soyoun Kim2,*, and Dong-Ki Lee1,*
1Global Research Laboratory (GRL) for RNAi Medicine, Department of Chemistry, Sungkyunkwan University (SKKU), Suwon 16419, Korea, 2Department of Bioengineering, Dongguk University, Seoul 04620, Korea, 3Department of Biomedical Engineering and School of Mechanical Engineering, Inje University, Gimhae 50834, Korea
Correspondence to:*Correspondence: dklee@skku.edu (DKL); skim@dongguk.edu (SK)
Keywords: aptamer, biolayer Interferometry, biosensor, cell-SELEX,
Food and water contamination by pathogenic bacteria is a serious threat to the human health. Therefore, fast, accurate and economic methods of detection are critical to the timely diagnosis of virulent strains in the infected sources (Koedrith et al., 2015). As many pathogens are not easily detected, indicator organisms are a fundamental monitoring tool to assess the microbiological status of water and food (Tortorello, 2003). Detection of safety indicators suggest the presence of conditions associated with increased risk of exposure to a pathogen. These, along with yeasts, molds and some other bacterial species, also include
Conventional methods of
Unless otherwise noted, all chemicals were purchased from Sigma Aldrich or invitrogen. SELEX library (N40 library) was obtained from IDT and transcribed using DuraScribe® T7 Transcription Kit (Epicentre Biotechnologies, USA) following the manufacturer’s protocol. Truncated aptamer Ec3(31), biotinylated or thiol modified oligonucleotides, HSR-1, SQ2 Mutant were chemically synthesized by ST pharma. Inc. Oligonucleotide sequence details are provided in Supplementary table 1. Lipoteichoic Acid (LTA) aptamer (OCT biotech, USA) was used as a positive control for gram positive bacteria and Lipopolysaccharide (LPS) aptamer (OCT biotech, USA) and
The starting 2′-F pyrimidine modified RNA library was prepared by
The screening of potential aptamers and calculation of binding affinity was done using qRT-PCR. The four selected aptamer sequences were
Truncated aptamer Ec3(31) with 3′ biotin, SQ2 mutant-biotin and N40 primer-biotin controls, were allowed to bind to 20 μl of streptavidin coated magnetic beads (dynabead, Invitrogen, USA) for 15 min at RT. yeast tRNA (0.1 mg/ml) and BSA (1 mg/ml) were added and the incubation was further continued for 45 min. Unbound aptamer were washed-off and this preblocked bead-aptamer complex were incubated with various cell density of
ForteBio’s Octet RED 384TM instrument and streptavidin (SA) sensors (Pall life sciences, USA) (Cat: 18-5019) were used to study interactions between aptamer candidates and bacteria cells or bacteria lysates, according to manufacturer’s instructions. All the aptamer target interactions were performed in binding buffer. In the case of antibody and control aptamers (LTA and LPS), 1X kinetic buffer (Fortebio) and buffer recipe provided by OTC biotech were used in accordance with the manufacturer’s protocol. All aptamer candidates were heated at 95°C, chilled on ice for 5 min, and suspended in binding buffer at RT prior to use. SA sensors were pre-wet by binding buffer for 30 min followed by loading with aptamer candidates at 25°C for 1200 s at 1000 rpm. After washing out nonspecific binding aptamers, sensors were blocked with biocytin and dipped into bacteria cells and lysates with desired concentrations. Association and dissociation profiles were measured for 10 min. To monitor non-specific binding for biosensor, a reference sensor (without aptamer loading) was performed with the same procedure as aptamer loaded sensors. Kinetic screening results were acquired by subtraction from the reference sensor using Octet data analysis software, version 7.0 (ForteBio). In the case of aptamer inhibition, SA sensors were pre-coated with biotinylated truncated aptamers and then associated with lysates and unlabeled aptamer mixtures (pre-incubation at RT for 20 min before BLI assay).
Two Cr/Au Gap capacitance electrodes of thickness 5 nm and 15 nm respectively were deposited on Pyrex glass (7740) by thermal evaporation process. The Cr/Au layer were patterned and etched as shown in Fig. 4A. Parylene was deposited at 2 μm thickness at each electrode and patterned to form flow channels. One electrode makes inlet and outlet holes for sample introduction and removal, and two parylene coated electrodes are finally bonded by thermo-compressing bonding (150°C, at 24 MPa for 20 min).
Thiol modified and Cy3 labeled Ec3 aptamer were mixed with 10 mM NaCl. Four concentrations of aptamer solution were tested for immobilization (40 pM, 0.4 nM, 4 nM and 18 nM) and incubated for 1 hour. The intensities of fluorescent signal, representing the aptamer immobilization capacity, were monitored. The unbound sample was washed by injection of PBST (0.1% tween 20 in PBS buffer). The chip was heated to 90°C and slowly cooled down to allow proper aptamer secondary structure formation. The chip was washed with DI water thoroughly to remove the unbound aptamer. 1 mM BSA solution was introduced into the channel and incubated for 30 min. Serial dilutions of bacteria solution (108, 106, and 104 CFU/ml) was introduced onto the chip. After 1 h of incubation, the chip was washed thoroughly and the bound bacteria were stained with Sybr Gold and visualized under fluorescent microscopy (Nikon eclipse TE2000-U). For impedance analysis, EIS measurement were performed which swept frequency from 1 Hz to 1 MHz under low applied voltage (less than 0.01 V) to avoid electrochemical reaction between electrode and liquid solution. As a negative control, we also measured impedance of buffer solution as a function of frequency.
12 rounds of selections were performed to obtain
In order to check the potential of aptamer Ec3 as an
Biolayer interferometry or BLI is an optical analytical technique that allows real-time monitoring of molecular binding events occurring on the surface of the biosensor (Concepcion et al., 2009). BLI has been shown to be an efficient label-free assay format to detect molecular interactions. (Kammer et al., 2014; Zichel et al., 2012). It analyzes the pattern of white light reflected from two surfaces: a layer of immobilized protein on the biosensor tip, and an internal reference layer. Any change in the number of molecules bound to the biosensor tip causes a shift in the interference pattern that can be measured in real-time. Use of BLI in detection of whole pathogens is a very novel approach and not many studies have been done in this regard. In a recent and probably the first study where BLI has been used to detect whole bacteria, gram-negative bacterium
To further improve upon the sensitivity of aptamer mediated
Nyquist plot from impedance spectroscopy of various concentrations of bacteria solution and negative control are given in Fig. 4C. Each impedance signal is clearly different to that of negative control (No bacteria). Impedance increases with bacterial concentration. (Supplementary Fig. 4). In low frequency range such as 1 Hz, impedance increase is caused by capacitance, since phase angle is close to - 90 deg. However, at higher frequency (over 1 kHz) resistance increases in proportion to the number of bacteria in solution. When frequency is over 10 kHz, impedance value is close to constant (Supplementary Fig. 4) and it increases in accordance with the density of bacteria concentration. It suggests that bacteria binding onto the electrode work as resistance particles, inhibiting current flow between electrodes. The system that we developed here can be used as a sensor to monitor
The 2′F modified RNA aptamer developed in this study has far better
Although our assay sensitivity is lesser in comparison to some of the impedance based detection of
. Aptamer sequences identified.
Sequence family | Aptamer sequence | % of total sequences |
---|---|---|
Ec3 | AUACCAGCUUAUUCAAUU | 20 |
Ec2 | AUACCAGCUUAUUCAAUU | 7 |
Ec5 | AUACCAGCUUAUUCAAUU | 6.7 |
Hye-Min Woo, Jin-Moo Lee, Chul-Joong Kim, Jong-Soo Lee, and Yong-Joo Jeong
Mol. Cells 2019; 42(10): 721-728 https://doi.org/10.14348/molcells.2019.0157Jihoon Kim, and Won Do Heo
Mol. Cells 2018; 41(9): 809-817 https://doi.org/10.14348/molcells.2018.0295Koen Van Laer, and Tobias P. Dick
Mol. Cells 2016; 39(1): 46-52 https://doi.org/10.14348/molcells.2016.2328