Description of Instrumentation and Procedures - Advances in Biochemical Engineering Biotechnology - page 8

with a chopper-stabilized operational amplifier. This bridge produced a maxi-
mum change of 100 mV in the recorder signal for a temperature change of
0.001 °C. However, the lowest practically useful temperature range was, limited
mainly by temperature fluctuations caused by friction and turbulence in the
column: typically 0.005-0.01°C. Differential temperature recordings were made
against a reference thermistor that was inserted in a pocket in the inner alumi-
num block or against an identical thermistor probe with an inactive reference
column. The latter arrangement was useful when nonspecific heat effects (e.g.,
due to heat of solvation or dilution) were encountered. The sample was then split
equally between the enzyme column and the reference column [18]. Alterna-
tively, the second channel could be reserved for another enzyme preparation,
permitting a quick change of enzymatic analysis. Some instruments were even
equipped with a dual Wheatstone bridge, enabling two different, independent
analyses to be carried out simultaneously. In such conventional systems it is an
advantage to replace used-up enzyme columns quickly, although the fresh
column requires about 20-30 min to achieve thermal equilibrium with the cir-
culating buffer. In total, over 30 instruments of the newer type have been assem-
bled at the work-shop of our centre for use in industry and in research institutes.
A major drawback has been the unwillingness of the public to accept an
uncommon technique, resulting in fewer developments. The common mode re-
jection (detection is non specific), non discriminative, compared to spectroscop-
ic (wherein specific wavelength is used) and electrochemical (wherein specific
potential is used) for detection. In addition the technique is not very sensitive.
The theoretical sensitivity is 0.1

M using the GOX/catalase system.
There are other more sensitive techniques, such as amperometry chemilumi-
nescence and fiber fluorimetry, that have recently been developed in our labor-
atory which are not as robust as the ET. The high concentration of enzyme
employed in the column may be treated both as an advantage and disadvantage.
This increases the operational stability of the column to over a couple of years,
compared to a few months with lower enzyme loads.The transducer (thermistor)
has no fouling or drift, thus making the calibration extremely simple. Such an
approach is useful for bioprocess monitoring and scaling up or down is simple.
The general approach can be used for a multiple enzyme system with thermistors
at the inlet and outlet of each enzyme block, and the carry-over heat - if any - can
be subtracted for each enzyme/substrate combination. Oxygen (for oxidase reac-
tions) can be regenerated by electrolysis of water, with the use of platinum elec-
trodes, directly in the flow stream in the vicinity of the enzyme. Similar approa-
ches had already been demonstrated by us. The pH changes - if any - are negli-
gible and well within the buffering capacity of the circulating buffer, and do not
pose a major problem while using multiple enzymes.

m

2.2
Minisystem

The design of mini and micro systems calls for a multidisciplinary approach
involving engineering, materials science, electronics and chemistry. With the
advances in integrated circuit technology and micromachining of liquid filters,