Nanofluidic Devices with 8 Pores in Series for Real-Time, Resistive-Pulse Analysis of Hepatitis B Virus Capsid Assembly.
To improve the precision of resistive-pulse measurements, we have used a focused ion beam instrument to mill nanofluidic devices with 2, 4, and 8 pores in series and compared their performance. The in-plane design facilitates the fabrication of multiple pores in series which, in turn, permits averaging of the series of pulses generated from each translocation event. The standard deviations (σ) of the pulse amplitude distributions decrease by 2.7-fold when the average amplitudes of eight pulses are compared to the amplitudes of single pulses. Similarly, standard deviations of the pore-to-pore time distributions decrease by 3.2-fold when the averages of the seven measurements from 8-pore devices are contrasted to single measurements from 2-pore devices. With signal averaging, the inherent uncertainty in the measurements decreases; consequently, the resolution (mean/σ) improves by a factor equal to the square root of the number of measurements. We took advantage of the improved size resolution of the 8-pore devices to analyze in real time the assembly of Hepatitis B Virus (HBV) capsids below the pseudocritical concentration. We observe that abundances of assembly intermediates change over time. During the first hour of the reaction, the abundance of smaller intermediates decreased, whereas the abundance of larger intermediates with sizes closer to a T = 4 capsid remained constant.
Department of Chemistry and ‡Department of Molecular and Cellular Biochemistry, Indiana University , Bloomington, Indiana 47405, United States.
Panagiotis
P
Kondylis
Department of Chemistry and ‡Department of Molecular and Cellular Biochemistry, Indiana University , Bloomington, Indiana 47405, United States.
Jinsheng
J
Zhou
Department of Chemistry and ‡Department of Molecular and Cellular Biochemistry, Indiana University , Bloomington, Indiana 47405, United States.
Zachary D
ZD
Harms
Department of Chemistry and ‡Department of Molecular and Cellular Biochemistry, Indiana University , Bloomington, Indiana 47405, United States.
Andrew R
AR
Kneller
Department of Chemistry and ‡Department of Molecular and Cellular Biochemistry, Indiana University , Bloomington, Indiana 47405, United States.
Lye Siang
LS
Lee
Department of Chemistry and ‡Department of Molecular and Cellular Biochemistry, Indiana University , Bloomington, Indiana 47405, United States.
Adam
0000-0001-9945-6267
A
Zlotnick
Department of Chemistry and ‡Department of Molecular and Cellular Biochemistry, Indiana University , Bloomington, Indiana 47405, United States.
Stephen C
0000-0003-2415-041X
SC
Jacobson
D002213:Capsid / Q000737:chemistry* / Q000378:metabolism; D006515:Hepatitis B virus / Q000737:chemistry* / Q000378:metabolism; D056656:Lab-On-A-Chip Devices; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation* / Q000379:methods; D058608:Nanopores; D019065:Virus Assembly