Concluding remarks

To our knowledge, this is the first study demonstrating a hybrid inorganic-organic nanomaterial designed with the purpose of mapping metabolites in microscale biological specimens by mass spectrometry. This novel strategy brings about several key advantages: (i) application of the hybrid matrix can be conducted in liquid phase (native environment of the biological cells), (ii) the matrix targets the biological specimen by attaching to its surface due to electrostatic interactions, (iii) application of the organic MALDI matrix compound occurs

in situ, following the attachment of nanoparticles, and the controlled-release step (in alkaline

vapors), which warrants less dispersion of analytes than using standard matrix application techniques, (iv) the expenditure of chemicals is minimized. Another aspect of this analytical strategy is related to labwork safety: The implementation of many standard chemistry and biochemistry procedures may be hazardous to researchers and pose threat to the environment, for example: carcinogenic ethidium bromide is frequently used as a DNA marker in gel electrophoresis, solvents such as tetrahydrofuran or chloroform are used in synthetic protocols despite their explosive nature, eventually – toxic matrices are sprayed using gas-powered painting guns in order to obtain MALDI images of biological specimens. The proposed imaging strategy – involving hybrid nanoparticles – can mitigate the exposure of the experimenters to toxic chemicals, and – at the same time – ensure excellent lateral resolution;

therefore, it addresses important issues related to MALDI imaging.

Chapter 3

Recording temporal characteristics of convection currents by continuous and segmented-flow sampling

3.1...Introduction

A vast majority of contemporary developments and applications in liquid-phase chemistry deals with homogeneous solutions. Organic chemists pay a lot of attention to adequate mixing of the reaction mixtures; inhomogeneities could result in a decreased reaction efficiency, or produce system instabilities. In analytical chemistry, homogenisation of samples is essential for ascertaining reliable and reproducible results. However, many natural and human-triggered processes are non-homogeneous; examples include the diffusion of atmospheric gases into ocean waters, and dispersion of environmental pollutants in the atmosphere, or in water reservoirs. Chemical gradients are omni-present in the macro-world and micro-worlds: for example, organisms emit signalling molecules into their habitats while individual cells do the same in their microenvironments in order to exert a response of other biological entities. Diffusion and convective mixing further contribute to the dispersion of these chemical species until an equilibrium is reached. On the sub-cellular and supra-cellular levels, chemical gradients are responsible for the evolution of important physiological processes.

Analytical methods for studying the propagation of chemical waves in non-equilibrated mixtures are scarce. For instance, when analyzing three-dimensional samples using

spectrophotometric tools, one has to assume a perfect homogeneity of a solution, so that the absorption laws are obeyed (cf. section 1.4). Therefore, spectrophotometry in its standard format is not directly applicable to the analysis of chemical heterogeneity. Same holds for the modern analytical techniques such as mass spectrometry (MS), in which case samples are typically loaded into syringes in order to infuse them via electrospray ionization (ESI) emitter into MS orifice; this normally eliminates the possibility of studying chemical gradients present in liquid media.

As outlined in section 1.5, Traditional sampling methods employing capillary tubing or microfluidic devices are likely to blur the spatiotemporal gradients due to advection and diffusion. Although Taylor dispersion of sample plugs transported in microfluidic channels can be reduced by implementing electrokinetic flow, electric fields are not always compatible with the samples studied. Sampling by segmented flow may readily be achieved by simultaneous injection of two immiscible fluids into a capillary or a microfluidic channel.^27,28 A considerable advantage of segmented flow is that one can reduce the dispersion of analytes as the discrete samples are transported along the flow line. In analytical protocols, segmented flow systems can readily be coupled with various types of analytical instruments; in fact, this approach has widely been used in enzyme assays,^60,61 protein analysis,^62,63 small molecule analysis,^32-34,64 and single-cell analysis.^65,66 Segmented flow can also be used in conjunction with various detection platforms, including nuclear magnetic resonance⁶⁷ and mass spectrometry (MS).^68-71

Building on the previous work, here we show two facile methods for recording spatiotemporal gradients formed during convection of substances in liquid media – used as a model process showing the dynamics of chemical heterogeneity in the three-dimensional space. The methods presented here are either based on continuous or segmented flow fluid transport, and used in conjunction with visible-range absorbance and/or mass spectrometric detection.

3.2...Materials and methods 3.2.1...Materials

Acetic acid, caffeine, ferroin, and n-octanol were purchased from Sigma-Aldrich (St Louis, USA). Blue ink (Simbalion, New Taipei City, Taiwan) was purchased from a local stationery shop. The fused silica capillary with the ID 150 μm (OD 375 μm) was purchased from GL Science (Tokyo, Japan), while the fused silica capillary with the ID 320 μm (OD 435 μm) and ID 450 μm (OD 673 μm) were purchased from Polymicro (Phoenix, USA). Tygon tubing was purchased from Saint Gobain (Akron, USA) and Idex (Oak Harbor, USA).

3.2.2...Construction of the flow-through optical detector

The multi-point tri-wavelength flow-through optical detector enables measuring the absorbance of sample plugs delivered in segmented or continuous flow (Figure. 3.1A and 3.1B). It comprises 8 photoresistors (diameter: ~ 5 mm; CdS type; resistance range: 8-20 kΩ;

purchased from a local electronics shop) and 8 tri-colour light emitting diodes (LEDs;

The optical cell was incorporated into a plastic casing with 8 holes with a diameter of 4.1 mm, spaced at 8 mm (Figure 3.1A, left panel). The inner surface of the casing was lined with aluminium foil. An infrared µs-laser engraving machine (Huahia Laser, Taipei, Taiwan) was used to create eight pinholes (diameter,

 450 μm) in the aluminium foil. The pinholes

were spaced in a way that they exactly overlapped with the centre of the capillary used as the optical cell. Polyimide coating was removed from a standard GC-type fused silica capillary

Figure 3.1 Multi-point multi-wavelength flow-through optical detector. (A) Assembly of the device. (B) Absorbance measurement in an individual channel. Note that only up to 3 channels were used in this study.

(length: 12 cm; ID 450 μm, OD 673 μm) – used as the optical cell – and the transparent section of the capillary was precisely aligned with the pinholes along the aluminium foil lining inside the plastic casing. The two parts of the plastic casing were joined together, so that the positions of the LEDs on one side matched the positions of the photoresistors mounted on the opposite side (Figure 3.1A, left panel). Smaller holes (diameter ~ 1 mm) were drilled in the plastic casing in order to increase air circulation (Figure 3.1A, right panel).

The optical cell assembly was installed inside a styrofoam box (inner dimensions: 24 × 22 × 10 cm, w/d/h). Three PC-type electric fans were also installed in the box to enhance air circulation, and to facilitate heat dissipation. The fused silica capillary was connected to the upstream and the downstream parts of the flow line with Tygon tubing (ID 0.38 mm, OD 2.2 mm).

The wavelength of the light emitted by the tri-colour LEDs was controlled by the relay

board (Denkovi Assembly Electronics, Byala, Bulgaria). An analog/digital data logger (ADC-20; resolution: 20 bits; input range:

 2.5 V; preset sampling rate: 61-183 ms

data-point^-1; Pico Technology, St Neots, United Kingdom) was used to record the electric potentials at the outputs of the photoresistor circuits. Both devices were connected to a computer via USB ports, and operated with appropriate software packages. The segmented-flow data were treated using a custom software written in Free PASCAL (version 1.0.10 2009/04/10; B. Gábor, P. Muller, P. Vreman); the algorithm automatically removed the features due to n-octanol segments, and preserved the features due to the water-based segments.

3.2.3...Assembly of the three-port Y-junction for generating segmented flow

The design of the segmented flow generator is shown in Figure 3.2: Two fused silica capillaries (length: 2.5 cm; ID 320 μm; OD 435 μm), connected with Tygon tubing (ID 0.25 mm, OD 2.07 mm), were inserted to another piece of Tygon tubing with an ID of 0.7 mm (OD 3.2 mm). The distance between the outlets of the two fused silica capillaries (inside the 0.7-mm ID section of Tygon tubing) was  5 mm. The resulting junction was then sealed with Epoxy glue (plastic steel Epoxy resin; PowerBon, New Taipei City, Taiwan), and after setting for  1 h, the whole assembly was ready for use. In order to produce segmented flow, water and n-octanol (immiscible phases) were delivered via the inlet ports (Figure 3.2).

3.2.4...Measurements using the system coupled with a mass spectrometer

Hyphenating different detection systems in order to attain orthogonal chemical information is an important area of analytical science. In this study, we also coupled the home-made multi-point multi-wavelength detector with a mass spectrometer, so as to enable simultaneous monitoring of spatiotemporal gradients by two detection systems. Careful optimization of the experimental setup was necessary in order to select the flow rates which are suitable for the studied process (convection), and optical as well as mass spectrometric

Figure 3.2 The design of the Y-junction used to generate segmented flow.

detection. Pumping too much sample towards the orifice of the mass spectrometer may invite contamination of the instrument. On the other hand, decreasing flow rate during the sampling will also decrease the temporal resolution of the method. By implementing the ―split-flow‖

approach, and adjusting backpressures in the flow line, it was possible to achieve workable conditions for this setup.

When using such a hyphenated system, several modifications had to be introduced to the convection chamber and the sampling probe: Two fused silica capillaries (length: 6 cm; ID 320 μm; OD 435 μm) were transferred through the septum in the cap (18-MSL-ST3; Thermo Fisher Scientific, Waltham, USA) of the 20-mL glass vial (20-HSV; Thermo Fisher Scientific) acting as the ―convection‖ chamber. One of these capillaries was connected to the syringe pump, and the other one was connected to the downstream flow line (Tygon tubing) with the optical detector and the mass spectrometer. The fused-silica capillary used as the ESI emitter (length: 2 cm; ID 150 μm; OD 375 μm) was mounted in a tiny hole made in the Tygon tubing (ID 0.38 mm). The distance between the ESI emitter and the orifice of the ion trap MS was relatively long ( 2 cm) in order to prevent contamination of the instrument when

concentrated samples were analyzed. Since the section of Tygon tubing mounted downstream from the ESI emitter exerted slight backpressure on the liquid in the flow line, a small portion of the liquid was diverted to the fused silica capillary section acting as the ESI emitter.

Recently, a method of introducing samples to mass spectrometers without the need for establishing an electrical connection at the emitter was published.⁷² In our system, we also skipped the electrical connection at the ESI emitter, which simplified the design and operation of the system without damaging its performance. In this study, we used the amaZon speed ion trap mass spectrometer from Bruker Daltonics (Bremen, Germany). The voltage applied to the ion transfer capillary was -5500 V (positive-ion mode), and the end-plate offset was set to 500 V. The flow rate of the dry gas was set to 12 L min^-1. The mass range was 70-380 Da, and the accumulation time was 0.5 ms.

At the beginning of the experiment, before acquiring data, the test analyte (ferroin) was injected into the lower part of the glass vial filled with

 21 mL of an aqueous solution

containing caffeine (4.8 × 10^-5 M) and acetic acid (0.5 %). The data acquisition started when the syringe pump was turned on, and 0.5%-solution of acetic acid was pumped into the vial.

Since the vial was completely filled with the liquid medium, the acetic acid solution – injected with the syringe pump – exerted hydrodynamic pressure on its contents; as a result, the liquid medium was pushed out via the second capillary mounted in the septum. This setup provided an adequate flow stability for the continuous MS analysis.

3.2.5...Measurement of the flow rate in the ESI emitter

In order to measure the effective flow rate of the sample in the ESI emitter, we implemented the experimental setup shown in Figure 3.3. Water was pumped along the flow line by syringe pump at the flow rate of 30 μL min^-1. Effluent was collected at the outlet of the Tygon tubing (length: 30 cm; ID 0.13 mm) during 10 min. The net weight of the effluent aliquots was determined using analytical balance. The procedure was performed with the

mass spectrometer on and off, and also after the removal of the ESI emitter. The flow rate of liquid sample in the ESI emitter was then calculated by subtraction of the flow rates determined for the eluate collected at the outlet of the Tygon tubing (Figure 3.3A) – without and with ESI emitter installed (Figure 3.3B).

Figure 3.3 Measurement of the effective flow rate in the fused silica capillary used as ESI emitter. (A) The ESI emitter is installed in the flow line, and the MS is on. (B) The ESI emitter is taken away, and adhesive tape is used to prevent leakage of the liquid medium through the small hole made in the wall of the Tygon tubing.

3.3...Results and discussion

3.3.1...Monitoring convection-driven currents by optical detector absorbance detection

Here we demonstrate probing chemical waves, which are formed in a liquid medium due to convection, by using continuous flow sampling in conjunction with optical absorption detection. A small amount of medium is sampled and transferred along the capillary flow line

towards detector by using either segmented flow or non-segmented (continuous) flow. At the beginning of the experiment, we injected a small aliquot of ferroin solution into the bottom of a 20-mL glass vial. Subsequently, the lower part of the vial was heated up ( 34 °C) in order to develop a temperature gradient, and induce convective mixing of the ferroin solution with the liquid medium present in the vial. We used a syringe pump operated in the withdrawal mode in order to pull the contents of the vial with the flow rate of 30 μL min^-1. n-Octanol was simultaneously injected to the Y-junction (installed along the flow line) by a syringe pump operated in the infusion mode at the flow rate of 6 μL min^-1 (Figure 3.4). The volume of each aqueous plug is estimated to 1.25 ± 0.16 (SD) μL.

Figure 3.4 Recording convection with segmented and continuous flow. (A) Experimental setup used in the real-time sampling with/without segmented flow prior to detection by the flow-through optical detector (cf.

Figure 3.1). The segmented flow was generated by pushing n-octanol towards the Y-junction while the bulk of the liquid was withdrawn by a syringe pump at the outlet of the flow line.

Figure 3.5 shows photographs of the inlet vial representing the convection-driven mixing of ferroin (red) with the liquid medium (transparent) while Figure 3.6 shows the corresponding raw data. The ―ups‖ and ―downs‖ in the original signal trace are caused by the differences in refractive indices and extinction coefficients of n-octanol and the aqueous samples. The relative heights of the lower section of the valleys in Figure 3.6 vary according

to the absorbance of the plugs sampled from the vial. In order to simplify the representation of the data, a custom software was used to remove the signal of n-octanol, and the data treated this way are displayed in Figure 3.7A (red line). From this data it is clear that the signal increased due to the increasing absorbance of ferroin. Interestingly, the increase of the absorbance with time (in relative units) cannot be described by any simple function, as one could do for the diffusion process. The gradual mixing of ferroin with the liquid medium is a chaotic process, and the trace in Figure 3.7A (red line) represents numerous fluctuations before the signal stabilizes at a level when the mixture became a homogeneous solution.

Figure 3.5 Photograph of the vial (nominal volume: 20 mL) during the convective mixing of 100 μL ferroin with 15 mL water (aided by heating). The ID of capillary used for on-line sampling was 320 µm.

Next, we carried out a similar experiment but using this time continuous (non-segmented) flow to transfer samples from the glass vial to the detector. This yielded curves which also represent some fluctuations (Figure 3.7B, red line). However, in this case, the traces are much smoother than the curves obtained using segmented flow (Figure 3.7A, red line). This unwanted ―smoothing effect‖ is due to advection and diffusion (e.g. ref. 73), taking place in the flow line – between the sample inlet and the detector. Compared with the continuous flow (Figure 3.7B), the segmented flow (Figure 3.7A) helps to preserve temporal

Figure 3.6 Convective mixing of ferroin with water followed by segmented flow and flow-through optical detector (cf. Figure 3.1; wavelength: 518 nm). The red line represents original data while the blue line shows the final data extracted by the custom software. The two traces were shifted vertically for clarity.

and spatial resolution of the digitized three-dimensional sample (cf. Figure 3.5).

In both cases, the traces can be fit with exponential functions (Figure 3.7A and 3.7B, blue dashed line), which represent the mixing trends. Apart from the fluctuations caused by convection currents, the equilibriums are reached at similar times (taking into account the small difference between the effective sampling flow rates in the segmented -flow and continuous flow systems). In Figure 3.8, the experimental data (cf. Figure 3.7A and 3.7B, top graphs, red line) were subtracted with the values predicted by the fitted exponential functions (cf. Figure 3.7A and 3.7B top graphs, blue dashed line): this representation highlights the presence of strong fluctuations of relative absorbance due to the convection process. These fluctuations are especially apparent in the middle of the data record, i.e. 400-800 s (segmented flow, Figure 3.8A), and 300-700 s (continuous flow, Figure 3.8B). From Figure 3.7A it is

Figure 3.7 Recording convection with segmented and continuous flow. (A) The output data (red line) obtained with the segmented flow sampling (2 replicates). The blue dashed lines correspond to the exponential functions fitted to the experimental data (after removal of n-octanol-related features from the trace): a: f(t)

= 31 × (1 – e^(-0.003t)); b: f(t) = 52 × (1 – e^(-0.003t)). The features marked with asterisks (*) are due to air bubbles. (B) The output data (red line) obtained with the continuous flow sampling (2 replicates). The blue dashed lines correspond to the exponential functions fitted to the raw data: a: f(t) = 27 × (1 – e^(-0.003t));

b: f(t) = 43 × (1 – e^(-0.003t)).

also clear that, at some points, the relative absorbance values (represented by the measured potentials) are much higher than the equilibrium absorbance at the end of the data record. This points out an important feature of convection current; unlike in diffusion, a momentary concentration of the substance in the three-dimensional space may be higher than the concentration of this substance after complete mixing of the substance with the medium. This feature has implications on the real-world convection systems, for example, the release of pollutants to the environment. Overall, the experiments discussed above show the feasibility of sampling convection-induced waves from liquid media on the scale of micro- to millilitres.

Figure 3.8 An alternative representation of the data sets displayed in Figure 3.7A and 3.7B (upper graphs). The experimental data points were subtracted with the fitted exponential functions (A: f₁(t) = 31 × (1 – e^(-0.003x)); B: f₂(t) = 27 × (1 – e^(-0.003x))). Fluctuations of absorbance due to convection currents in the glass vial can be clearly seen.

In order to realize the possibility of performing measurements at various wavelengths, offered by the detector described above (cf. Figure 3.1), we further attempted the monitoring of sequential convection of substances with different absorption maxima (Figure 3.9). First, an aliquot of 100 µL of a blue ink was injected into to the lower part of the 20-mL glass vial, which was then heated up to induce convection, and segmented flow was then used for sampling (Figure 3.9A). The blue ink absorbs green and red light (wavelength range of red:

600-700 nm, green: 490-560) but it does not absorb blue light (wavelength range 450-490 nm,

cf. Figure 3.10). Therefore, one could observe the fluctuating increase of the signal in the

detection channels operating at wavelengths 629 and 518 nm (Figure 3.9A). Subsequently, a 100-µL aliquot of ferroin was injected into the lower part of the same vial. This time the detection channels operating at wavelengths 518 and 463 nm produced a significant change in the light absorption traces (Figure 3.9B).

3.3.2...Monitoring convection-driven currents by optical detector and mass spectrometer

Optical detection benefits from the direct dependence of absorbance on concentration, warranted by the Beer-Lambert law (equation 3, page 8); however, it has poor selectivity. In order to demonstrate the possibility of continuous sampling of chemical waves – such as those due to the convection currents – we have attempted coupling the home-made flow-through optical detector with a mass spectrometer. Several technical problems had to be solved to assure satisfactory performance of the system: One way to improve the temporal resolution of the sampling system is to increase the sampling rate. However, when coupling fluidic systems with MS, it is not desirable to work with high flow rates. Therefore, we have implemented split-flow coupling of the sampling setup with MS via an electrode-free ESI interface. A section of Tygon tubing with a relatively small ID (0.13 µm) was used to produce back

Optical detection benefits from the direct dependence of absorbance on concentration, warranted by the Beer-Lambert law (equation 3, page 8); however, it has poor selectivity. In order to demonstrate the possibility of continuous sampling of chemical waves – such as those due to the convection currents – we have attempted coupling the home-made flow-through optical detector with a mass spectrometer. Several technical problems had to be solved to assure satisfactory performance of the system: One way to improve the temporal resolution of the sampling system is to increase the sampling rate. However, when coupling fluidic systems with MS, it is not desirable to work with high flow rates. Therefore, we have implemented split-flow coupling of the sampling setup with MS via an electrode-free ESI interface. A section of Tygon tubing with a relatively small ID (0.13 µm) was used to produce back

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