Hendrik J. Viljoen

Hendrik J. Viljoen 

Hendrik J. Viljoen
Contact Information:
211 OTHM
Lincoln: City Campus
(402) 472-9318
hviljoen1@unl.edu
Email   
Personal Links:
Professional Memberships:

Distinguished Professor and Department Chair
  • Ph.D., University of Pretoria, South Africa, 1985
  • M.S., University of Pretoria, South Africa, 1981
  • B.S., University of Pretoria, South Africa, 1979

Areas of Research and Professional Interest
  • Development of new methods for the synthesis and processing of materials
  • Analysis of complex engineering systems with the aid of models and simulation
  • Application of chemical reaction engineering principles to interdisciplinary problems. The following areas are of particular interest:
    • Mechanical-Chemical interaction in solid phase reactants
    • Cooperative behavior or mesoscale: solitons and solitary wave
    • Thermal stresses and crack development in chemically reacting system
    • Development of high temperature acoustic detection methods
    • Piezoelectric sensors, manufacture and modeling
    • Chemical reaction and natural convection
    • Analysis of thermal CVD systems, e.g. - the effect of the flow field on the deposition rate and film homogeneity
    • Dynamic stress development of films during deposition
    • Reaction-induced thermal stresses in consolidated media
    • Mechanical failure of catalytic systems during thermal cycling or transient operation
    • The study of nonlinear dynamical systems, e.g. - existence of temporal chaos and solitary waves
    • Bifurcation analysis of non-catalytic highly exothermic reacting systems
    • Development of fast thermocyclers for polymerase chain reaction (PCR)
    • Theoretical investigation of errors in the polymerase chain reaction process
    • Assembly of DNA structures from synthetic oligonucleotides
    • Strategies for Gene Packaging in Virus-Like Particles
Inventions/Patents
  • Whitney, S.E. and H.J. Viljoen, “Sample Tube”, U.S. Pat. D 659,848 issued May 15, 2012
  • Viljoen, H.J. and S.W. Whitney, “Laboratory Analysis Device Housing” U.S. Pat.  D 665, 917 issued August 21, 2012.
  • Viljoen, H.J. and Jooste, B.R., "Piezoelectric Sensors/Actuators for use in refractory environments," U.S. Patent 6,057,628, issued May 2, 2000
  • Whitney, S., Viljoen, H.J., Padhye, N. and Nelson, M.R., "A gas jet process and apparatus for high-speed amplification of DNA." PCT Patent, issued in 2005 in 22 countries in Europe and Asia
Courses Taught

About Hendrik J. Viljoen

Research


  • Point-of-care diagnostics of TB in resource poor settings*

    A technology is developed to diagnose tuberculosis at low cost in resource poor settings. The diagnostics consists of the following steps: sputum samples are collected, DNA is extracted from bacilli in the sputum, target regions on the DNA are amplified by the polymerase chain reaction (PCR) and products are detected optically by fluorescent molecular beacons. The total turn-around time of the process is less than sixty minutes. Minimal human intervention is required during the process to reduce infection of health care workers and lessen the demand on skilled staff. The design is modular: the first module is a sputum processing cassette that performs 4 functions: (1) chemical modification of the sputum's rheology to promote flow and mixing, (2) lysis of bacilli to release DNA (3) removal of PCR inhibitors from sputum and (4) transfer of processed sputum to PCR well (also part of the cassette). The second module is a custom-built rapid thermocycler that houses the cassette. PCR amplification of DNA in sputum samples takes 6 to 10 minutes for 30 cycles and that time includes hybridization of targets to molecular beacons.

    The Mycobacterium tuberculosis complex (MTC) consists of M. tuberculosis, M. bovis, M. africanum and M. microti. Members of the complex are identified by insertion sequences, IS6110 and IS1081.

    Custom Thermocycler


    Gel electrophoresis
    Typical output of our custom thermocycler using molecular beacons for insertion sequence IS1081. Forty cycles were completed. The protocol allows 2 seconds per cycle for molecular beacon/template hybridization.
        Gel electrophoresis results of IS1081 and IS6110 of H37Rv in PCR volumes of 100 ml and 150 ml. All reactions consist of 30 cycles, a 30sec. hot start and a 25 sec. final elongation. Using a no-hold (for elongation) protocol and a rapid polymerase, the 150 ml reaction was completed in 6 minutes. The more conservative protocol took 10 minutes


    Besides speed, the custom thermocycler has the unique feature to PCR amplify variable volumes at the same rate. Volumes between 25 and 150 ml are processed at the same speed. The ability to use more sputum in nucleic acid amplification reactions increases sensitivity.

    *In this context point-of-care (POC) settings refer to peripheral clinics where smear microscopy is performed. These clinics offer minimal infrastructure; refrigeration and electricity (grid or generators).

  • Biomolecular engineering:

    The application of reaction engineering principles to molecular biology problems, has led to novel insight and discoveries. Kinetic models have been developed for the polymerase chain reaction (PCR) to describe the editing and proof-reading capabilities of the enzyme, the average mutation frequencies and the effects of dexoynucleotide pool compositions and temperature on elongation and error rates. Quantitave models have been developed to assess the errors which accrue during a PCR reaction. Two sources of errors are associated with the PCR process: (1) editing errors that occur during DNA polymerase-catalyzed enzymatic copying and (2) errors due to DNA thermal damage. The errors which are ascribed to the polymerase depend on the efficiency of its editing and proof-reading functions and are closely related to the kinetic models we have developed. Thermally induced errors stem mostly from three sources: A+G depurination, oxidative damage of guanine to 8-oxoG and cytosine deamination to uracil. The post-PCR modifications of nucleic acids at elevated temperatures are more pronounced if the DNA is in a single-stranded form. Below melting temperatures, the DNA molecules are mostly in the double stranded form and the hydrolytic attack on the DNA bases is sterically hindered. Two hydrolytic damage reactions are prominent at elevated temperatures; C deamination and A+G depurination.

    The kinetic and error models are combined to simulate PCR experiments and to provide the following measures: (1) yield (2) error frequency (3) optimum reaction conditions. The software resides on the fast thermocyclers we use in our laboratory. The rapid thermocyclers perform typical PCR experiments in 3 to 5 minutes and lend themselves not only to rapid diagnostics, but also to any PCR application that warrants low thermal damage.

    PCR Thermometer
    Click image to view video clip of typical temperature-time profiles of our rapid PCR thermocycler. (Windows Media 1.4MB)

    The combination of rapid PCR and low thermal damage creates an advantage for assembling synthetic oligonucleotides into large DNA molecules by PCR. The PCR assembly technique has been used to synthesize a large number of genes with exceptional accuracy and speed. All eight genes of the Influenza A virus have been synthesized and together with collaborators from Austria, the genes have been packaged into virus like particles (VLP). The dual approach of experiments and theoretical modeling is also followed in PCR assembly. Theoretical models have led to optimal assembly strategies. Typical assembly times for 2,000 base pair structures are 15 to 20 minutes. The current generation of rapid thermocyclers is modified for PCR assembly to automatically capture synthesized parts and ligate them into final constructs.

    VLP
    Electron microscope picture of VLP that contains synthetic HA gene.

    As part of the effort to optimize the expression of synthetic genes in cells, a new theoretical project on codon efficiency has started. It is called the tri-frame coding theory. The genetic code uses all three reading frames to encode for the following information: what must be synthesized, how much of it must be synthesized and how accurately it must be synthesized. The zero reading frame (0RF) encodes for the amino acid sequence. The combination of rare codons in the 0RF and stop codons in the 1RF controls the ribosome transit time of the mRNA and hence the expression level. Rare codons in 0RF causes the ribosome to frame-shift and the incomplete polypeptide chain is tagged and terminated in the -1RF and +1RF with high probability. If the out-of-frame stop frequency is low, termination is delayed and ribosome processing time is extended and vice versa. Entropy is the antithesis of accuracy. Codon definition in the DNA is presumably exact and thus the (information) entropy is zero. Mistranscription causes an increase in the codon's entropy. A further entropy increase follows the translation step. The system's entropy is the weighted sum of codon entropies and the probability distribution of the ribosome's occupancy of the reading frames. The tri-frame coding theory provides exact expressions for: (1) the yield of error-free protein, (2) the fraction of prematurely terminated polypeptides, (3) the percentage mistranscription in proteins, (4) the percentage mistranslation in proteins, (5) the percentage mutations due to frameshifting and (5) the energy and entropy cost to synthesize a protein.

    Reaction engineering of heterogeneous systems The research is focused on solid phase reactants. In an effort to increase the rate of reaction of metal/oxide mixtures, the general approach has been to reduce the particle sizes of reactants. It has also been recognized that heating by compression (as in a shock wave) is much faster than thermal conduction and some studies have been undertaken by other investigators to explore the possibility to ignite pyrotechnic mixtures by impact, but in most cases the conversion was not complete and most reaction occurred in the post-shock region with little or no contribution to the shock wave. Research on mechano-chemical reactions is conducted with the use of a Bridgman anvil. The project is multi-disciplinary and the following subdivisions are addressed.

    Probability distribution
    Probability distribution of ribosomal occupancy of the three reading frames of mRNA of the rpsU gene of Escherichia coli. The step-reduction at codon 23 in the zero reading frame (0RF) is associated with the rare codon Cysteine. Out-frame stop codons terminate ribosome occupancy in the +1 and -1 RF

    (1) Physical properties of mixtures
    (2) Kinetics of metal/oxidizer mixtures
    (3) Mechano-chemical reactions
    (4) Post-reaction behavior

    In 1935, Bridgman reported results of combined hydrostatic pressure and shear for a wide variety of materials. Whilst most substances undergo polymorphic transformation, some react violently: PbO decomposes quiescently to a thin film of lead while PbO2 detonates yielding a residue of Pb. Reactive mixtures Al/Fe2O3 and Al/Bi2O3 produce even stronger mechanical-chemical interaction.

    PCR Thermometer
    Click image to view video clip of thermite reaction in our Bridgman anvil. (Windows Media 380k)

    A theory has been developed to determine the contact area between heterogeneous mixtures. The contact area is influenced by the presence of pores and this effect has been included in the model. Based on this theory, more realistic kinetic models have been developed for solid/solid reactants. The theory has also been used to develop more advanced models of transport properties, such as effective thermal conductivity, susceptibility, permeability and electrical resistivity. The contact theory proves to be quite useful to describe heterogeneous effects such as Kapitza resistance and impedance mismatch.

    The kinetics of metal/oxidizer mixtures have been measured with the ETA-100 instrument (developed by Dr. Alexander Shteinberg). The instrument allows the user to register and record transient temperatures based on either the brightness or the color of the sample surface during electro-thermal explosion. After dry mixing the mixture is pressed to obtain a cylindrically-shaped specimen measuring 10-12 mm long and 3 mm in diameter. An electric current is sent through the sample until it reaches a pre-set temperature. The current is switched off at that point and further temperature rise is due to chemical reaction alone. Chemical reaction already starts during the preheating stage, albeit small. The temperature of the electro-thermal analyzer is measured by an array of optical diodes at intervals of 0.625 mm along the length of cylindrical specimen. An array of sixteen optical diodes tracks the temperature on the cylinder surface at a sampling rate 10,000 data points per second for each diode. The data are stored and analyzed to determine the kinetic parameters.

Honors and Awards

  • Editorial Board of Computational Biology & Chemistry, from 2006
  • 2005 Multidisciplinary Research Award, University of Nebraska-Lincoln
  • Research on rapid polymerase chain reaction reviewed in Nature: Moore, P., (2005) "PCR: Replicating Success." Nature 435; 235-238
  • Co-Author of the section, "Modeling of Chemical Reactors", for Ullman's Technical Encyclopedia, new edition
  • Co-Chair of sessions on nano-energetic materials at AIChE annual meetings in 2004 (Austin), 2005 (Cincinnati)
  • Chair of session at CHISA 2004, Prague, Czech Republic (micro-reactors), International SHS Conference, Cagliari, Italy (2005) (modeling of solid phase combustion)
  • Chair and co-chair of session on combustion synthesis at AIChE annual meetings in 1989 (San Francisco), 1990 (Chicago), 1991 (Los Angeles), 1992 (Miami), and 1993 (St. Louis), 1999 (Dallas), 2000 (Los Angeles)
  • Guest editor of a special issue of Combustion Science Technology {88} (1992)
  • 1990 Researcher of the Year Award in the Faculty of Engineering, University of Stellenbosch
  • 1989 Presidents Award for excellence in research
  • 1980 Silver Medal of South African Institute of Chemical Engineers

Selected Publications

  • Grobler, A, O. Levets, S. Whitney, C. Booth and HJ Viljoen, “Rapid Cell Lysis and DNA Capture in a Micro Lysis Reactor”, Chem. Eng. Sci. 81 pp311-318. (2012)
  • Freifeld, A, Simonsen, K.A., Booth, C.S., Zhao, X, Whitney, S.E., Karre, T., Iwen, P.C. and HJ Viljoen, “A new rapid method for Clostridium difficileDNA extraction and detection in stool:  toward point-of-care diagnostic testing” J. Mol. Diag. 14 (3) pp274-279. (2012)
  • Louw, T, H.J. Viljoen, S. Whitney and A. Subramanian, “Forced Wave Motion with Internal and Boundary Damping”, J. Appl. Phys. 111 no 014702. (2012)
  • Pienaar, E. and H.J. Viljoen, “A State-Time Epidemiology Model of Tuberculosis” Comp. Biol. & Chem. 36 pp15-22. (2012)
  • Guha Thakurta, S., Viljoen, H.J. and A. Subramanian, “Evaluation of the real-time protein adsorption kinetics on albumin-binding surfaces by dynamic in situ spectroscopic ellipsometry” Thin Solid Films  520 (6) pp2200-2207 (2012)
  • Louw, T., C. Booth, E. Pienaar, J. TerMaat, S. Whitney, and H. Viljoen. “Experimental Validation of a Fundamental Mathematical Model for PCR Efficiency.” Chemical Engineering Science. 66  pp1783-1789 (2011).
  • Whitney SE, Louw TM  and Viljoen HJ, “Optimization of Oligonucleotide Design” AIChE J 57 pp1912-1918.(2010).
  • Booth CS, Pienaar E, Termaat JR,  Whitney SE, Louw TM  and Viljoen HJ, “Efficiency of the Polymerase Chain Reaction” Chem. Eng. Sci. 65 pp4996 – 5006 (2010).
  • Pienaar E, Fluitt A, Whitney SE, Freifeld, AG and Viljoen HJ,  “ A Model of Tuberculosis Transmission and Intervention in an Urban Residential Area.” Comp. Biol. & Chem. 34 pp86-96 (2010).
  • Valente WJ, Pienaar E, Fast A,  Fluitt A, Whitney SE, Fenton RJ, Barletta RG, Chacon O, Viljoen HJ,  “A Kinetic Study of In Vitro Lysis ofMycobacterium smegmatis.” Chem. Eng. Sci. 64 pp1944-52 (2009).
  • Pienaar E, Whitney SE,  and Viljoen HJ, “A model of the complex response of Staphylococcus aureus to methicillin.” J. Theor. Biol. 257 pp438-445 (2009).
  • Mammedov T, Pienaar E, Whitney SE, TerMaat JR, Carvill G, Goliath R, Subramanian A, and Viljoen HJ, “A Fundamental Study of the PCR Amplification of GC-Rich DNA Templates.” Comp. Biol. & Chem. 32  pp452-457 (2008).
  • Pienaar E and Viljoen HJ "The Tri-Frame Model" J. Theor. Biol. 251: pp616-627 (2008). Pubmed# 18237749
  • Fluitt, A, Pienaar E and Viljoen, HJ, “Ribosome kinetics and aa-tRNA competition determine rate and fidelity of peptide synthesis”, Comp. Biol. Chem. (2007).
  • Tarlan G Mamedov, T.G., Kotera C, Padhye, N., Viljoen, H.J. and A. Subramanian “Rational “de novo” Synthesis of Genes by Rapid Polymerase Chain Assembly (PCA):  Assembly and Expression of Endothelial Protein-C Receptor Genes.” J. Biotech. (2007).
  • Griep, M.A., Kotera, C.A, Nelson, R.M. and Viljoen, H.J. “Kinetics of the DNA Polymerase Pyrococcus Kodakaraensis.” Chem. Eng. Sci. 61 pp3885-3892 (2006).
  • Pienaar, E., M. Theron, M. Nelson and H.J. Viljoen, “A Quantitative Model of Error Accumulation During PCR Amplification.” Comp. Biol. & Chem. 30 pp102-111. (2006).
  • Griep M, Whitney S, Nelson M and Viljoen HJ “DNA Polymerase Chain Reaction: A Model of Error Frequencies and Extension Rates”  AIChE J. 52 pp384-392 (2006).

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