Sensitivity enhancement of affinity-based electrical biosensor using pulsed measurement

국문 초록 (Abstract)

전기적 바이오센서의 최근 발전에도 불구하고, 광학적 방법에 비해 낮은 감도와 선택도는 실용화에 가장 큰 장애요소이다. 친화도 기반의 바이오센서에 있어서 나쁜 선택도는 낮은 친화력과 비특이 반응에 기인한다. 또한 특히 전기적 바이오센서에, 낮은 감도는 반대이온의 전하 차단 효과에 의한 전기적 신호의 약화에 기인한다.
본 논문에서는 전기적 바이오센서의 장점을 이용하여 선택도 및 감도를 동시에 향상시키는 펄스 측정 방법을 제안한다. 펄스 측정을 구현하기 위해 탄소 나노튜브 채널과 금 나노입자 위에 고정화된 프로브 DNA 및 방사형 전극으로 구성된 연구실에서 직접 제작된 센서 어레이를 사용하였고, 펄스 발생기와 증폭기로 측정 회로를 구성하였다. 상보적인 탐지대상 DNA 를 가한 후 그로 인한 채널 전도도의 변화를 통해 DNA 혼성화 정도 및 여부를 감지한다.
프로브와 대상 분자 사이의 혼성화 효율 향상을 위해 혼성화 중에 채널을 통해 프로브 분자에 전기적 펄스를 가하였다. 전하를 띄고 있는 프로브 분자는 전기 펄스에 의해 진동하며, 이는 DNA 혼성화 효율 향상을 이끌어낸다. DC 조건 및 전기를 가하지 않은 경우와 비교할 때, 펄스를 가하는 방법은 Tris-EDTA (TE) 완충용액에서의 실시간 측정에서 향상된 선택도로 비약적으로 감도 향상시켰다. 또한 혈청 조건에서도 상당히 낮은 농도의 한계까지 탐지할 수 있었다.
이온에 의한 전하 차단 효과를 극복하기 위해 계단 펄스에 대한 과도응답 관찰을 제안하였다. 과도상태에서는 계단 펄스에 의해 전기이중층이 순간적으로 확장하며, 그에 따라 전하를 가진 분자를 감싸던 반대 이온들이 밀려나게 된다. 단일가닥 DNA 와 이중가닥 DNA 의 과도응답을 비교하여, DNA 센서의 감도향상을 수치해석적 시뮬레이션과 실험으로 증명하였다. 과도상태 측정방법은 실시간 측정이나 또는 혼성화가 끝난 이후에도 적용 가능하다.
이 펄스 측정 방법은 DNA와 단백질, 펩티드 등의 탐지에 쓰이는 일반적인 전기적 바이오센서 플랫폼으로 확장 적용이 가능할 것이다. 본 연구를 통하여 우리는 펄스 측정 플랫폼과 결합된 전기적 바이오센서가 충분한 감도와 신뢰성으로 label 없는 대상 분자의 실시간 감지를 현실화시킬 수 있을 것으로 기대한다.

다국어 초록 (Multilingual Abstract)

Despite of recent advances in the electrical biosensors, lower sensitivity and worse selectivity than the optical sensors are the obstacles to success in practical uses. In the affinity-based biosensor including the electrical sensor, the worse selectivity results from the low affinity constant and activated non-specific binding event. Specifically for the electrical biosensors, low sensitivity arises from weakening of the electrical signal from the molecule charges due to the screening effect by the counter-ions.
In this dissertation, we propose a new scheme, hereafter the pulsed bias scheme, to enhance both the selectivity and the sensitivity in the electrical biosensor. In order to verify the scheme, we applied the pulsed bias to the biosensor platform developed by our group in Seoul National University, where the sensor cell arrays consist of the carbon nanotube network (CNN) channel formed on the concentric electrodes. The CNN channel is decorated with the Au (gold) nano particles (GNP) on which the thiolated probe DNA (p-DNA) immobilized. The island electrode in the concentric electrodes is connected to the pulse generator and the operational amplifier. From the change of channel conductance after applying complementary target DNA (t-DNA), we detect the DNA hybridization event.
For the enhancement of the hybridization rate between the probe and target molecule, electrical pulse bias is applied to the probe molecule via CNN channel during the DNA hybridization events. The probe molecule which has electrical charge oscillates with the pulse trains resulting in the enhancement of DNA hybridization efficiency. Compared with the DC and non-biasing condition, the pulse biasing method gives better selectivity and remarkable sensitivity enhancement in Tris-EDTA (TE) buffer solution in the real time measurement. In addition, very low limit of detection (LOD) can be achieved even in human serum condition.
To overcome the charge screening effect, we propose a transient measurement, where the measurement of the channel current is performed during the transient response of the mobile ions in the electrolyte system after the step-pulse bias applied to the drain electrode. On the transient state, electric double layer (EDL) is extended by the step-pulse bias instantaneously, and counter-ions screening the charged molecule are swept away. Comparing the transient response of between the single-stranded DNA (ssDNA) and the double-stranded DNA (dsDNA), we obtain the improved sensitivity in the measurement as well as in the numerical simulation results. The transient scheme may be applied during the hybridization event while the pulse bias is applied (real time measurement) or after completion of the hybridization events.
The pulsed bias scheme may be extended to general electrical biosensor platforms for detecting DNAs, proteins and peptides. The electrical biosensor platforms with the pulsed bias scheme may facilitate the label-free real time detection of the target molecule with considerable sensitivity and more reliable and rapid detection even in human serum.

목차 (Table of Contents)

• Abstract i
• Contents iv
• List of Tables vii
• List of Figures viii
• Chapter 1 1
• Introduction 1
• 1.1 Motivation 1
• 1.2 Selectivity issue 3
• 1.3 Sensitivity issue 4
• 1.4 Outline of the Dissertation 5
• Chapter 2 7
• Instrumentation for the pulsed bias to the electrical biosensor 7
• 2.1 Introduction 7
• 2.2 Sensor device preparation 8
• 2.2.1 Self-gating effect 9
• 2.2.2 Biosensor array fabrication 11
• 2.2.3 Immobilization of probe DNAs 14
• 2.3 DNA sensing in the CGi platform with DC measurement 15
• 2.3.1 Sensing mechanism 16
• 2.3.2 Real time measurement under the DC bias 17
• 2.4 Configuration for pulsed measurement 21
• 2.4.1 Composition of the pulsed measurement system 21
• 2.4.2 RC time constant of the sensor system 23
• 2.4.3 Separation of channel conductance from total output signal 24
• Chapter 3 29
• Selectivity enhancement by pulse biasing during DNA hybridization 29
• 3.1 Motivation for enhancement of affinity kinetic parameters 30
• 3.1.1 Probe-Target binding 30
• 3.1.2 ka modulation by the pulsed bias condition 31
• 3.2 DNA hybridization under pulse biasing in TE buffer 34
• 3.2.1 Modulation of the DNA hybridization under pulse biasing 34
• 3.2.2 Real time measurement during DNA hybridization 36
• 3.2.3 Sensitivity after completion of DNA hybridization 40
• 3.2.4 Extraction of ka 43
• 3.3 DNA hybridization in serum under pulsed bias 46
• 3.4 Conclusion 49
• Chapter 4 50
• Pulsed measurement of the transient state for sensitivity enhancement 50
• 4.1 Introduction 50
• 4.2 Scheme of transient measurement 52
• 4.3 Simulation of electric double layer 54
• 4.3.1 Governing equations and simulation conditions 54
• 4.3.2 Simulation results for the steady state 57
• 4.3.3 Simulation results for the transient state 60
• 4.4 Transient measurement after step pulse bias 66
• 4.5 Conclusion 68
• Chapter 5 69
• Maximization of the sensitivity in the pulsed measurement configuration 69
• 5.1 Introduction 69
• 5.2 Scheme of the sensitivity maximization 70
• 5.3 Effect of hybridization enhancement in DC measurement 71
• 5.4 Additional sensitivity enhancement by the transient measurement 72
• 5.5 Conclusion 73
• Chapter 6 74
• Conclusion 74
• 6.1 Summary 74
• 6.2 Future works 75
• References 77