1
Biomimetic synthesis of silica nanospheres depends on the aggregation and phase separation of polyamines in aqueous solution

Type: Conclusion | Advantage: None | Novelty: None | ConceptID: Con1

2
Long-chain polyamines extracted from the highly siliceous cell walls of diatoms are known to precipitate silica nanospheres from aqueous, silicic-acid containing solutions at near-neutral pH in vitro.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac1

3
The same is true for synthetic polyamines such as polyallylamine.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac1

4
In the present contribution we show that the microscopic phase separation of polyallylamine in aqueous solution is strictly correlated with the silica precipitation activity of polyallylamine/silicic acid solutions.

Type: Conclusion | Advantage: None | Novelty: None | ConceptID: Con2

5
Multivalent anions such as phosphate or sulfate efficiently induce this microscopic phase separation.

Type: Conclusion | Advantage: None | Novelty: None | ConceptID: Con3

6
At higher anion concentrations, macroscopic phase separation occurs.

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs1

7
In contrast to the multivalent phosphate and sulfate ions, the monovalent chloride ions are much less efficient in polyallylamine aggregate formation.

Type: Conclusion | Advantage: None | Novelty: None | ConceptID: Con4

Introduction

8
Diatom cell walls are outstanding examples for nanoscale structured materials in nature.1

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac2

9
They consist of an amorphous composite material containing silica as well as certain biomolecules.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac2

10
The latter are assumed to play a crucial role in diatom cell wall formation.

Type: Motivation | Advantage: None | Novelty: None | ConceptID: Mot1

11
Examples for such biomolecules are the so-called silaffins isolated from the diatom Cylindrotheca fusiformis2 as well as the long-chain polyamines extracted from the cell walls of species such as Nitzschia angularis, Stephanopyxis turris, and others.3

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac3

12
Both these classes of molecules were also shown to precipitate silica from aqueous solutions containing silicic acid in vitro.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac1

13
The precipitates formed by the long-chain polyamines from S. turris consist of nanospheres exhibiting a surprisingly narrow size distribution.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac4

14
The particle diameter was shown to be strictly controlled by the concentration of phosphate ions added to the solutions.4

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac4

15
Initial NMR and dynamic light scattering experiments carried out on long-chain polyamines isolated from S. turris have shown that these molecules self-assemble in aqueous solutions.4

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac4

16
Silaffins from C. fusiformis behave very similarly.5

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac4

17
It is tempting to speculate that the aggregates (droplets) formed by the biomolecules in solution act as templates during cell wall formation.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac5

18
A model based on phase separation processes could be developed which explains the pattern formation in diatom cell walls.6

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac5

19
Inspired by these observations, a number of synthetic amino acids, peptides, and polyamines were shown to precipitate silica from aqueous solutions as well.7–11

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac6

20
New opportunities for the so-called biomimetic silica synthesis are likely to arise from these discoveries.

Type: Motivation | Advantage: None | Novelty: None | ConceptID: Mot2

21
One particularly interesting example is polyallylamine (PAA), a commercially available compound.

Type: Motivation | Advantage: None | Novelty: None | ConceptID: Mot2

22
It exhibits a long-chain structure similar to the polyamines found in diatoms.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac7

23
Polyallylamine was also shown to catalyze the polycondensation of silicic acid8 and to precipitate silica.9

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac7

24
The topic of the present paper is the detailed physico-chemical characterisation of PAA in aqueous solution especially with respect to the question whether or not an aggregation of the PAA molecules is a prerequisite for their silica-precipitating function as it is the case for the above-mentioned biomolecules extracted from diatoms.

Type: Goal | Advantage: None | Novelty: None | ConceptID: Goa1

Experimental

Materials

25
Polyallylamine hydrochloride (PAA) was purchased from Aldrich.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp1

26
The PAA molecule exhibits a repeated unit (ru) of the following structure: [–CH2CH(CH2NH2˙HCl)–]n with n ∼ 160 corresponding to a molecular weight of ca. 15 kDa.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp1

NMR and dynamic light scattering experiments

27
NMR spectra were acquired on a DMX-500 spectrometer (Bruker, Karlsruhe, Germany) operating at 500 MHz 1H resonance frequency.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp2

28
The calibration of the 31P NMR spectra was performed using H3PO4 (85%) as an external reference.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp3

29
Dynamic light scattering (DLS) experiments were carried out on a MALVERN HPPS5001 nanosizer.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp4

30
For the NMR and dynamic light scattering analyses, the polyallylamine hydrochloride was dissolved in millipore water.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp5

31
A concentration of 1 mM PAA was chosen.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp5

32
The desired concentration of orthophosphate ions (Pi) was obtained by the addition of the corresponding amount of sodium dihydrogen phosphate to the samples.

Type: Method | Advantage: None | Novelty: New | ConceptID: Met1

33
The final pH value was adjusted to 5.8 by the addition of NaOH.

Type: Method | Advantage: None | Novelty: New | ConceptID: Met2

34
Sulfate containing samples were prepared by the addition of sodium sulfate and chloride containing samples by the addition of sodium chloride.

Type: Method | Advantage: None | Novelty: New | ConceptID: Met3

35
Before the measurements, all solutions were allowed to equilibrate for at least 2 days.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp6

In vitro precipitation of silica and SEM analysis

36
For the silica precipitation experiments, a freshly prepared solution of 1 M tetramethoxysilane in 1 mM HCl was incubated at 20 °C for 15 min and immediately used as a source of monosilicic/disilicic acid.12

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp7

37
A typical precipitation assay contained in 50 µl: 0.2 mM polyallylamine and phosphate (5 to 25 mM).

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp8

38
Silica formation was initiated by the addition of 2 µl silicic acid, prepared as described above.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp9

39
After 12 min at 20 °C, precipitated silica was collected by centrifugation (4 min, 12.000 rpm).

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp10

40
The precipitate was washed twice with water, then suspended in water, applied to an aluminium sample holder, and air dried.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp11

41
Silica precipitates were analyzed without sputter-coating with a LEO1530 field-emission scanning electron microscope equipped with energy dispersive X-ray analysis (EDXA, Oxford Instruments).

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp12

42
Colorimetric determination of silica precipitates was performed by the molybdenum blue method12 after dissolving precipitated silica in 20 µl 2 M NaOH (85 °C, 5 min).

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp13

Results and discussion

The influence of phosphate ions

43
Fig. 1 summarizes the results of silica precipitation experiments carried out for samples containing various amounts of phosphate.

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs2

44
The amount of silica precipitated from the PAA/silicic acid solution is plotted as a function of the phosphate concentration.

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs2

45
Silica precipitation takes place only above a threshold value of ca. 0.3 [Pi]/[ru].

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs2

46
Below, no silica precipitates at all indicating a crucial role of the phosphate ions.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res1

47
It is interesting to note in this context that the PAA-induced silica precipitation experiments described by Patwardhan et al9. were carried out in a phosphate-containing buffer solution.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac8

48
Furthermore, the particle diameter of the silica nanospheres precipitated by PAA is correlated with the phosphate concentration in complete analogy to the observations made for the long-chain polyamines extracted from S. turris.4

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac8

49
The maximum particle diameter of ca. 3 µm strongly exceeds the maximum value of about 1 µm obtained for the polyamines from S. turris.4

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac8

50
A possible explanation for this behaviour is the much smaller size of the polyamines from S. turris (1.55 kDa molecular weight).

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac8

51
On the other hand, the particles precipitated by the synthetic polyallylamine exhibit a much broader size distribution than the nanospheres obtained by using polyamines from S. turris.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac8

52
In order to understand this behaviour, we have studied aqueous PAA solutions of various phosphate concentrations by dynamic light scattering (see Fig. 2).

Type: Goal | Advantage: None | Novelty: None | ConceptID: Goa2

53
In the absence of phosphate, aggregates of an average diameter of about 1 nm could be detected.

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs3

54
For a single PAA molecule, one would expect a diameter of the same order of magnitude.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res2

55
This leads to the conclusion that the PAA molecules exist as single molecules (monomers) or small clusters in the absence of phosphate ions.

Type: Conclusion | Advantage: None | Novelty: None | ConceptID: Con5

56
Addition of phosphate results in a continuous but slow increase of the aggregate diameter up to about 5 nm at 0.25 [Pi]/[ru].

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs4

57
Note, that this phosphate concentration is close to to the above-mentioned threshold value of ca. 0.3 [Pi]/[ru] necessary for silica precipitation.

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs4

58
A rapid increase of the aggregate diameter is observed beyond this threshold value.

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs5

59
At 0.31 [Pi]/[ru], aggregates of about 120 nm diameter could be detected.

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs6

60
Visual inspection shows that the samples become cloudy at this concentration which confirms the existence of larger aggregates (see Fig. 3).

Type: Result | Advantage: None | Novelty: None | ConceptID: Res3

61
If the phosphate concentration is further increased, the samples remain cloudy up to about 0.5 [Pi]/[ru].

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs7

62
DLS measurements show that the aggregate diameter increases up to almost 600 nm at 0.44 [Pi]/[ru] while a decreased value could be found at 0.5 [Pi]/[ru].

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs8

63
This decrease can be explained as follows: 1H NMR studies show that more than 2/3 of the polyamine has disappeared from the part of the sample inserted into the NMR tube.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res4

64
Obviously, this amount of polyamine already sediments at the bottom of the sample without forming a visible separate phase.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res4

65
The decreased concentration of polyamine in the top part of the sample must be responsible for the decreasing diameter of the aggregates.

Type: Conclusion | Advantage: None | Novelty: None | ConceptID: Con6

66
At 0.63 [Pi]/[ru] and higher concentrations, the samples become clear after equilibration but exhibit a macroscopic phase separation: A separate phase containing the polyamine is formed at the bottom of the tube.

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs9

67
The aqueous phase on top does no longer contain a detectable amount of polyamine aggregates.

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs10

68
The observed aggregation and phase separation can be explained by the attractive electrostatic interactions between the positively charged polyamine molecules and the negatively charged phosphate ions13 and/or the formation of hydrogen bonds.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res5

69
Comparison of Figs. 1 and 2 shows that the formation of the described large polyamine aggregates, i.e., microscopic phase separation is correlated with the capability of the system to precipitate silica from a PAA/silicic acid solution.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res6

70
If, however, macroscopic phase separation takes place, i.e. for phosphate concentrations significantly exceeding 0.5 [Pi]/[ru], the solution looses its silica precipitation activity.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res7

71
That means, microscopic phase separation is a necessary prerequisite for silica precipitation by the polyamine solution.

Type: Conclusion | Advantage: None | Novelty: None | ConceptID: Con7

72
In order to characterize the physico-chemical properties of this microscopically phase separated solution in more detail, extended 31P and 1H NMR spectroscopic studies were carried out.

Type: Object | Advantage: None | Novelty: New | ConceptID: Obj1

73
For all samples, only one single 31P signal is observed.

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs11

74
The chemical shift, δ, as well as the linewidth, Δν1/2, (full-width-at-half-maximum) of this signal, however, change.

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs12

75
The chemical shift is plotted as a function of phosphate concentration in Fig. 4 (top).

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs13

76
It remains almost constant up to about 0.3 [Pi]/[ru].

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs14

77
Beyond this phosphate concentration, δ continuously decreases.

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs15

78
At phosphate concentrations higher than 0.6 [Pi]/[ru], that means in the range of macroscopic phase separation, the chemical shift is again nearly constant and amounts to ca. 1.2 ppm.

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs16

79
As the solution in the NMR tube is almost completely free of polyamine at high phosphate concentrations, the latter chemical shift value represents free phosphate ions.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res8

80
From the observation that only a single signal is observed for all concentrations it is concluded that phosphate ions interacting with the polyamine rapidly exchange with free phosphate ions (on the time scale of NMR spectroscopy).14,15

Type: Conclusion | Advantage: None | Novelty: None | ConceptID: Con8

81
The increased and constant chemical shift at low phosphate concentrations indicates that phosphate is preferentially bound to the polyamine molecules up to about 0.3 [Pi]/[ru].

Type: Result | Advantage: None | Novelty: None | ConceptID: Res9

82
Above this concentration, an increasing amount of free phosphate exists which results in the described decrease of chemical shift.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res10

83
The linewidth of the 31P NMR signals (Fig. 4, bottom) also exhibits an interesting behaviour.

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs17

84
From an initial value of about 4 Hz it increases up to about 12 Hz for 0.4 [Pi]/[ru].

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs17

85
At higher concentrations, Δν1/2 decreases until a constant value of 1.6 Hz is found for the aqueous part of the macroscopically phase-separated samples.

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs18

86
This behaviour can be explained as follows: Due to the rapid exchange between phosphate bound to the polyamine aggregates and free phosphate, an average linewidth is observed.14,15

Type: Result | Advantage: None | Novelty: None | ConceptID: Res11

87
It is known from the chemical shift analysis that the phosphate ions are preferentially bound to the polyamine aggregates at low phosphate concentrations.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res12

88
The linewidth, therefore, corresponds to the linewidth in the polyamine-bound state for low phosphate concentrations.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res13

89
Since an increasing aggregate diameter (see Fig. 2) results in a lower correlation time of rotational diffusion, an increasing linewidth is expected for increasing phosphate concentrations.

Type: Hypothesis | Advantage: None | Novelty: None | ConceptID: Hyp1

90
This is in agreement with the experimentally observed behaviour up to 0.4 [Pi]/[ru] (Fig. 4, bottom).

Type: Result | Advantage: None | Novelty: None | ConceptID: Res14

91
For concentrations above 0.4 [Pi]/[ru], the influence of the increasing amount of free phosphate is observed.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res15

92
Since the linewidth of free phosphate is smaller than that of polyamine-bound phosphate, the measured average linewidth then decreases with increasing phosphate concentration.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res15

93
After macroscopic phase separation took place, only free phosphate exists in the NMR samples giving rise to the small and constant linewidth of 1.6 Hz observed for high phosphate concentrations.

Type: Conclusion | Advantage: None | Novelty: None | ConceptID: Con9

The influence of sulfate and chloride ions

94
The influence of sulfate and chloride ions upon aqueous solutions of PAA was also investigated.

Type: Object | Advantage: None | Novelty: New | ConceptID: Obj2

95
The behaviour of sulfate, another multivalent anion, turned out be very similar compared to phosphate: The solutions become cloudy at about 0.3 [sulfate]/[ru] and exhibit macroscopic phase separation above 0.5 [sulfate]/[ru].

Type: Result | Advantage: None | Novelty: None | ConceptID: Res16

96
This behaviour shows that the ability to phase-separate aqueous PAA solutions must not be considered to be a specific property of phosphate ions.

Type: Conclusion | Advantage: None | Novelty: None | ConceptID: Con10

97
It is striking that both, phosphate and sulfate are multivalent anions.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac9

98
The question arises whether or not monovalent anions such as chloride can also be used to induce the described effects.

Type: Object | Advantage: None | Novelty: New | ConceptID: Obj3

99
Fig. 5 shows the diameter of the polyamine aggregates determined by dynamic light scattering as a function of chloride ion concentration.

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs19

100
Obviously, aggregates of increasing diameter are observed at increasing chloride concentrations.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res17

101
However, even for the highest chloride concentration (ca. 20 [Cl]/[ru]), the obtained aggregate diameter of ca. 9 nm is almost two orders of magnitude smaller than the maximum aggregate diameter of ca. 600 nm observed for a phosphate concentration of only 0.44 [Pi]/[ru].

Type: Result | Advantage: None | Novelty: None | ConceptID: Res18

102
It is furthermore important to note that the macroscopic phase separation found for phosphate and sulfate does not occur for chloride ions even at about 20 [Cl]/[ru].

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs20

103
That means, the monovalent chloride ions are much less efficient in attracting the PAA molecules compared to the multivalent phosphate and sulfate ions.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res19

104
In agreement with these observations, no silica is precipitated at all from the chloride-containing PAA solutions even at chloride concentrations up to ca. 20 [Cl]/[ru] which is far beyond the physiologically relevant level.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res20

105
In summary, it can be stated that microscopic phase separation is necessary for the PAA-induced silica precipitation from silicic-acid containing aqueous solutions.

Type: Conclusion | Advantage: None | Novelty: None | ConceptID: Con11

106
This microscopic phase separation can be induced by the addition of multivalent anions such as phosphate or sulfate to the aqueous solutions.

Type: Conclusion | Advantage: None | Novelty: None | ConceptID: Con12

107
Polysilicic acid molecules may be adsorbed on and/or dissolved in the polyamine microdroplets thereby forming a coacervate which hardens by silica formation.

Type: Conclusion | Advantage: None | Novelty: None | ConceptID: Con13